STRUCTURAL INSIGHT INTO MICROBIAL NATURAL PRODUCT PATHWAYS
By
KUNHUA LI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Kunhua Li
To my Family
4
ACKNOWLEDGMENTS
Graduate school has been a wonderful time for me. There are so many people
whom I wish to thank during my PhD study at the University of Florida. I would most
certainly not be writing this dissertation without all the support.
I would like to offer my sincere thanks to my advisor and committee chair, Prof.
Steven Bruner. Steve, I consider myself to be extremely lucky to be a member of the
Bruner lab. I enjoy your mentorship style. Your patience and trust in me have fostered
me as an independent researcher and allowed me to accomplish things that I often
thought I would not be able to do. You have been, and still will be the role model for me.
I believe our supervisor-student relationship will continue to be a lifelong intellectual
friendship.
I also thank my other committee members Prof. Rebecca Butcher, Prof. Nicole
Horenstein, Prof. Robert McKenna, and Prof. Wei Wei. I greatly appreciate your time
reviewing this work. To Prof. Horenstein, you have been a source of encouragement,
always willing to discuss science, teaching as well as academia life and career with me.
I would like to thank Prof. McKenna for all the insightful discussions and suggestions on
X-ray protein crystallography.
I would like to thank all our collaborators. I would like to thank Prof. Butcher in the
AcoX project and Prof. Adrian Roitberg in the DpgC project. I wish I had enough room to
include these interesting and promising projects in my dissertation. To Prof. Andrew
Hansen, I appreciate your help on the MmuM project. You assisted me to go through
the HMT manuscript line by line, which greatly improved my writing. I also appreciate
your resourceful discussions in other metabolites repair projects. To Prof. Yousong
5
Ding, I enjoyed the collaborations on Y3 and other projects. And personally, I am
exceedingly grateful for your instructions and recommendations in my job-hunting.
Lastly, I would like to thank the staff on the Brookhaven National Laboratory and
Argonne National Laboratory for X-ray access, without whom I can barely finish any of
my projects.
To my fellow colleagues and friends, I would thank Dr. Heather Condurso. I came
here as one of the first Bruner Lab members in UF, and you certainly helped us a lot,
like a big sister. Now, as a senior graduate student myself, I can really appreciate your
kindness and patience back in the days. Dr. Jarrod Mousa, Wei-Hung Chen, Aleksandra
Zagulyaeva, Matthew Burg, Brian MacTavish and other Bruner members, you provided
me your advice and friendship that have made my graduate experience (and my
bachelors party!) wonderful. Gengnan Li, it is my great pleasure to work with you, and
thank you for your help in several projects. To Dr. Rachel Jones and Dr. Zhanglong Liu,
you provide me with valuable advises and suggestions on my career. To Xinxing Zhang
of the Butcher Lab and Dr. Guang Yang, Peilan Zhang, Yi Zhang of the Ding Lab, I
enjoyed working with you in the past years.
My family is my lifelong support. My father Jinfu Li and mother Peiyu Zhang
encouraged me to come to the state for graduate school, and I never regretted about it.
My wife Jingwen Liu, you know I will say a thousand thanks for all your support and
understanding. I am so lucky to have you in my life. The work I present in the following
pages is dedicated to them.
6
I would like to thank all the funding support to my projects, include the grants
from NSF, NIH and UF (to S.D.B.). I thank the assistantship from UF chemistry. UF
CLAS Dissertation Fellowship also supports this dissertation.
7
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES .......................................................................................................... 11
LIST OF FIGURES ........................................................................................................ 12
LIST OF ABBREVIATIONS ........................................................................................... 15
ABSTRACT ................................................................................................................... 20
CHAPTER
1 MICROBIAL SIDEROPHORE-BASED IRON ACQUISITION AND MEDICINAL APPLICATION ........................................................................................................ 22
1.1 Introduction of Microbial Siderophores .............................................................. 22
1.2 General Approaches of Siderophore Biosynthesis ........................................... 23
1.3 Siderophore Secretion ...................................................................................... 26
1.4 Holo-Siderophore Acquisition ........................................................................... 28
1.5 Siderophore Utilization ...................................................................................... 30
1.6 Siderophores as Therapeutical Drug Leads ...................................................... 31
1.7 Targeting Siderophore Pathways ...................................................................... 34
1.8 Conclusions and Insights .................................................................................. 35
2 STRUCTURE AND MECHANISM OF THE SIDEROPHORE INTERACTING PROTEIN FROM THE FUSCACHELIN GENE CLUSTER OF Thermobifida fusca ....................................................................................................................... 37
2.1 Siderophore Utilization Pathway in Thermobifida fusca .................................... 37
2.2 FscN is a Flavin Containing Siderophore Interaction Protein ............................ 40
2.2.1 FscN Contains a Non-covalently Bound FAD Cofactor ........................... 40
2.2.2 The Siderophore Utilization Protein FscN Binds Ferric-Fuscachelin ....... 41
2.2.3 FscN Specifically Reduces Ferric Fuscachelin ........................................ 42
2.2.4 Structure Determination with X-ray Crystallography ................................ 43
2.2.5 Proposed NADH Cofactor Binding Site in FscN ...................................... 47
2.2.6 Metal Binding Site Adjacent to the Flavin Cofactor .................................. 48
2.3 Structure of FscN Facilitate the Understanding of Siderophore Pathway ......... 49
2.4 Experimental Procedures .................................................................................. 57
2.4.1 General Methods ..................................................................................... 57
2.4.2 Cloning, Expression, and Purification of FscN ......................................... 57
2.4.3 FAD Determination, Reduction and Enzyme Kinetics.............................. 59
8
2.4.4 Purification of the Ferric-Fuscachelin Complex ....................................... 60
2.4.5 Purification Ferric-Enterobactin ............................................................... 60
2.4.6 Metal Analysis ......................................................................................... 61
2.4.7 Isothermal Titration Calorimetry Binding Determination .......................... 62
2.4.8 Coexpression of FscN-FscP and Western Blot Analysis ......................... 62
2.4.9 Crystallography, Structure Determination, and Refinement ..................... 63
3 STRUCTURE AND FUNCTIONAL ANALYSIS OF THE SIDEROPHORE PERIPLASMIC BINDING PROTEIN FROM THE FUSCACHELIN GENE CLUSTER OF Thermobifida fusca .......................................................................... 65
3.1 Periplasmic Binding Protein Facilitates Siderophore Delivery ........................... 65
3.2 Characterization of FscJ as a Siderophore Binding Protein .............................. 67
3.2.1 FscJ and Fuscachelin Delivery ................................................................ 67
3.2.2 FscJ Structure Determination with X-ray Crystallography ....................... 69
3.2.3 FscJ Ferric-Siderophores Interaction ....................................................... 72
3.3 Structural Features and Siderophore Interaction Mechanism ........................... 74
3.3.1 FscJ Structural Comparison with Other Type III PBPs ............................ 74
3.3.2 pH-Dependent Dynamics Observed in Crystal Structures ....................... 75
3.3.3 FscJ Has a Large Binding Pocket with Unique Charge Arrangement ..... 76
3.3.4 Conclusions and Insights ......................................................................... 79
3.4 Materials and Methods ...................................................................................... 79
3.4.1 Cloning, Expression and Purification of FscJ .......................................... 79
3.4.2 Cloning of Full Length FscJ, Expression and Localization ....................... 81
3.4.3 Isothermal Titration Calorimetry Binding Affinity Determination ............... 81
3.4.4 FscJ Crystallization and Optimization ...................................................... 82
3.4.5 FscJ Atomic Structure Determination and Refinement ............................ 83
3.4.6 Detailed FscJ SAD-MR Based Experimental Phasing ............................. 84
3.4.7 Modeling the FscJ/Ferric-Siderophore Interaction ................................... 85
3.4.8 FscJ Siderophore Binding Site Mutagenesis ........................................... 85
4 PRECURSOR PROTEIN-DIRECTED PEPTIDE MACROCYCLIZATION IN A RIBOSOMAL PEPTIDE NATURAL PRODUCT BIOSYNTHETIC PATHWAY ........ 87
4.1 RiPPs Biosynthesis and Microviridin Biosynthetic Pathway .............................. 87
4.2 Leader Protein-Directed Microviridin J Biosynthesis ......................................... 93
4.2.1 MdnB and MdnC Catalyze MdnA Cyclization .......................................... 93
4.2.2 MdnA Leader Peptide Interacts with MdnB and MdnC Macrocyclases ... 94
4.2.3 Overall Structure of MdnC and MdnB ...................................................... 98
4.2.4 Key Residues for Nucleotide Binding and Catalysis .............................. 105
4.2.5 Recognition of MdnA Leader Peptide by MdnC ..................................... 106
4.3 Structures of MdnC and MdnB Facilitate to Understand the Precursor Recognition in RiPP Pathway ............................................................................ 109
4.4. Materials and Methods ................................................................................... 115
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4.4.1 Protein Cloning, Expression and Purification ......................................... 115
4.4.2 MdnC Mutagenesis................................................................................ 117
4.4.3 Designed MdnA Variants ....................................................................... 117
4.4.4 Macrocyclization of MdnA Variants........................................................ 117
4.4.5 MdnC-MdnA Dicyclization Kinetics ........................................................ 117
4.4.6 Leader Peptide Binding Affinity Determination ...................................... 118
4.4.7 Preparation of SeMet Labelled Protein .................................................. 118
4.4.8 Crystallization, Data Collection and Crystallographic Analysis .............. 119
5 CRYSTAL STRUCTURES OF Y3 IN FUNGUS Coprinus comatus REVEAL A NOVEL LECTIN FAMILY WITH ANTIVIRAL AND ANTITUMOR ACTIVITIES ..... 122
5.1 Introduction of Lectins, and Proteinous Natural Product Y3 ............................ 122
5.2 Characterization of Y3 as a Novel Lectin ........................................................ 124
5.2.1 Genetic and Biochemical Characterization of Y3 .................................. 124
5.2.2 tNCS Complicates the Structure Determination .................................... 128
5.2.3 Structure of Y3 Reveals a Novel Lectin Family ..................................... 129
5.2.4 Carbohydrates Binding Site ................................................................... 131
5.2.5 Y3 with Antitumor and Antiviral Activities ............................................... 133
5.3 Discussions and Insights ................................................................................ 134
5.4 Materials and Methods .................................................................................... 138
5.4.1 Heterologous Expression of Mature Y3 ................................................. 138
5.4.2 Ellman's Test for Free Thiol Determination............................................ 139
5.4.3 Carbohydrate Content Determination .................................................... 139
5.4.4 Carbohydrates Specificity Determination with ITC ................................ 140
5.4.5 Protein Mass Analysis ........................................................................... 140
5.4.6 Crystallization of Y3 ............................................................................... 140
5.4.7 Diffraction Data Collection and Processing ........................................... 141
5.4.8 Atomic Structure Determination and Refinement .................................. 142
5.4.9 Models of Oligosaccharides Bound Y3 .................................................. 143
5.4.10 MTT Assay .......................................................................................... 143
6 HOMOCYSTEINE METHYLTRANSFERASE MmuM FROM Escherichia coli FACILITATES L-METHIONINE BIOSYNTHESIS AND DAMAGED COFACTOR (R,S)-S-ADENOSYL-L-METHIONINE REPAIR .................................................... 145
6.1 Metabolite Damage is an Under-Recognized Fact of Life ............................... 145
6.2 Homocysteine S-Methyltransferase and Metabolite Repair ............................ 145
6.3 HMT MmuM in E. coli ...................................................................................... 148
6.3.1 Biological Functions and Distribution of bacteria HMT .......................... 148
6.3.2 Overall Structure of MmuM in E. coli ..................................................... 150
6.3.3 The Zn2+-binding, Active Site ................................................................. 153
6.3.4 Modeling MmuM Substrate Recognition and Binding ............................ 157
6.3.5 Specific Potassium Ion Requirement for MmuM Activity ....................... 161
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6.4 Conclusions and Insights ................................................................................ 161
6.5 Materials and Methods .................................................................................... 162
6.5.1 Chemicals .............................................................................................. 162
6.5.2 Cloning, Expression, and Purification of MmuM .................................... 162
6.5.3 MmuM Crystallization ............................................................................ 163
6.5.4 Data Collection and Processing, and Structure Refinement .................. 164
6.5.5 Models of Bound Methyl-Donors and Analysis ...................................... 165
6.5.6 MmuM Active Site Mutagenesis ............................................................ 165
6.5.7 ITC-Based Activity Assay ...................................................................... 166
6.5.8 Determination of Protein Secondary Structure ...................................... 166
LIST OF REFERENCES ............................................................................................. 168
BIOGRAPHICAL SKETCH .......................................................................................... 195
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LIST OF TABLES
Table page
2-1 X-ray crystallography statistics of FscN .............................................................. 45
2-2 ICP-MS metal analysis of FscN .......................................................................... 49
2-3 PCR primers used for FscN related cloning ....................................................... 63
3-1 FscJ crystallization data collection and processing ............................................ 68
3-2 Protein structure comparison using the Dali server ............................................ 73
3-3 FscJ MR-SAD phasing and building cycles ........................................................ 84
3-4 Primers for FscJ site-directed mutagenesis ........................................................ 85
4-1 MS profiles of MdnA and MdnA variants ............................................................ 95
4-2 ITC-based precursor peptide/cyclase interaction ................................................ 98
4-3 Data collection and refinement statistics macrocyclases .................................. 102
4-4 Key residues in nucleotide interaction .............................................................. 103
4-5 Oligonucleotides for protein cloning and mutagenesis ..................................... 116
5-1 X-ray data collection, processing and structure refinement of Y3. .................... 127
5-2 Heavy atom derivative crystal preparation of Y3 .............................................. 142
6-1 X-ray data collection, processing and structure refinement of MmuM .............. 149
6-2 Kinetic parameters for HMT MmuM .................................................................. 155
6-3 Docking statistics of metallated MmuM and different methyl donors ................ 158
12
LIST OF FIGURES
Figure page
1-1 Microbial siderophores with diverse scaffolds..................................................... 23
1-2 Typical siderophore pathway in Gram-negative bacteria .................................... 25
1-3 Examples of siderophore related therapeutic drug leads. ................................... 32
2-1 The fuscachelin siderophore pathway of T. fusca............................................... 38
2-2 Annotated gene list of the T. fusca fuscachelin siderophore gene cluster .......... 39
2-3 Properties of the cofactor flavin bound FscN ...................................................... 41
2-4 ITC analysis of ferric-fuscachelin A binding to FscN ........................................... 42
2-5 FscN reduces ferric ion with the presence of NADH ........................................... 43
2-6 X-ray crystal structure of FscN ........................................................................... 44
2-7 Global multiple-protein sequence alignment of putative SIP proteins ................. 46
2-8 Two views of the flavin binding site .................................................................... 47
2-9 Domain arrangement of the flavoprotein reductase and electron transferase .... 54
2-10 S-Tag and His-tag Western blot for FscP-FscN coexpression............................ 56
3-1 FscJ purification and characterization ................................................................ 69
3-2 Overall structure of FscJ ..................................................................................... 70
3-3 Siderophore binding pockets in the SBP family .................................................. 71
3-4 ITC binding analysis of FscJ against purified ferric-fuscachelin A complex ........ 73
3-5 FscJ sequence alignments with clustalW2 ......................................................... 75
3-6 Molecular docking of FscJ with ferric fuscachelin A ............................................ 78
4-1 Representative precursor peptides and gene clusters in microviridin biosynthetic pathways ........................................................................................ 88
4-2 Biosynthesis of microviridin J.............................................................................. 91
4-3 Alignment of MdnC, MdnB and other reported ATP-grasp ligases ..................... 92
13
4-4 Expression and purification of MdnB and MdnC as dimeric proteins .................. 93
4-5 Analysis of full-length MdnA cyclizations ............................................................ 94
4-6 Kinetic analysis of the MdnC catalyzed dicyclization .......................................... 94
4-7 ITC profiles of MdnA variants interact with macrocyclases MdnB and MdnC ..... 96
4-8 Overall structures of MdnC and MdnB ................................................................ 97
4-9 Crystallographic packing of MdnC and MdnB ..................................................... 99
4-10 Electrostatics map of MdnC and MdnB dimers ................................................. 100
4-11 MdnC crystallized as a dimer............................................................................ 101
4-12 C-alpha distance difference plotting between MdnC and MdnB protomers. ..... 101
4-13 MdnC uses ATP to catalyze the macrocyclization ............................................ 103
4-14 Characterization of determinants for MdnC catalyzed cyclization..................... 104
4-15 MdnC central domain interacts with the precursor peptide MdnA..................... 104
4-16 Binding and cyclization activity of macrocyclases toward MdnA variants. ........ 107
4-17 MdnA variants and their macrocyclizations ...................................................... 107
4-18 Leader peptide directed peptide macrocyclization in the microviridin J biosynthetic pathway ........................................................................................ 114
5-1 Recombinant Y3 expression and characterization ............................................ 124
5-2 Characterization of purified Y3 ......................................................................... 125
5-3 tNCS complicates the structural determination of Y3 ....................................... 126
5-4 Overall structure of Y3 dimer ............................................................................ 128
5-5 High-resolution crystal structure of Y3 .............................................................. 132
5-6 Y3 interact with carbohydrates in a novel mechanism ...................................... 133
6-1 HMT converts L-homocysteine to L-methionine with unique substrate selectivity .......................................................................................................... 146
6-2 Overall structure of E. coli MmuM ..................................................................... 150
14
6-3 MmuM apo and metallated form crystals have different crystallographic packing ............................................................................................................. 151
6-4 MmuM belongs to the HMT superfamily of proteins ......................................... 152
6-5 The Zn2+ binding active site of MmuM .............................................................. 154
6-6 MmuM catalyzed kenetics ................................................................................ 155
6-7 Stability of MmuM mutants ............................................................................... 156
6-8 MmuM substrate binding and recognition ......................................................... 157
6-9 Crystallization and optimization of the MmuM .................................................. 163
15
LIST OF ABBREVIATIONS
A Adenylation Domain
ABC ATP-Binding Cassette
ACN Acetonitrile
ACP Acyl Carrier Protein
AdoHcy S-Adenosyl-L-Homocysteine
AdoMet S-Adenosyl-L-Methionine
ADP Adenosine Diphosphate
AMP Adenosine Monophosphate
Amp Ampicillin
Amx Amoxicillin
ANL Argonne National Laboratory
APS Advanced Photon Source
ASP Ammonium Sulfate Precipitation
AT Acyl Transferase
ATP Adenosine Triphosphate
BHMT Betaine Homocysteine Methyltransferase
C Condensation Domain
CD Circular Dichroism
CHES 2-(Cyclohexylamino)ethanesulfonic Acid
CoA Coenzyme-A
16
Cy Cyclization Domain
DH Dehydratase
DHB 2,3-Dihydroxybenzoic Acid
DMSO Dimethyl
DTT Dithiothreitol
E Epimerization Domain
EDTA Ethylenediaminetetraacetic Acid
ESI Electrospray Ionisation
FAD Flavin Adenine Dinucleotide
FPLC Fast Protein Liquid Chromatography
FSR Ferric-Siderophore Reductase
Hcy L-Homocysteine
HEPES 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic Acid
HMT Homocysteine S-Methyltransferase
HPLC High-Performance Liquid Chromatography
ICP-MS Inductively Coupled Plasma-Mass Spectrometry
IMAC Immobilized Metal Affinity Chromatography
ITC Isothermal Iitration Calorimetry
KR Ketoreductase
KS Ketosynthase
LC-MS Liquid Chromatography–Mass Spectrometry
17
LCP Lipid Cubic Phase
LS-CAT Life Sciences Collaborative Access Team
LSP Lipid Sponge Phase
MAD Multi-Wavelength Anomalous Dispersion
MALDI-TOF Matrix Assisted Laser Desorption Ionization Time-of-Flight
MDR Multidrug Resistance
MES 2-(N-morpholino)ethanesulfonic Acid
MetS Methionine synthase
MFS Major Facilitator Superfamily
MIC Minimum Inhibitory Concentration
MIR Molecular Replacement
MMT Methionine S-methyltransferase
MPD Methyl-2,4-Pentanediol
MR Multiple Isomorphous Replacement
MS Mass Spectrometry
Mt Methylation Domain
MTHF 5-Methyltetrahydrofolate
NADH Nicotinamide Adenine Dinucleotide
NADPH Nicotinamide Adenine Dinucleotide Phosphate
NBD Nucleotide-Binding Domains
NIS NRPS-Independent Synthetase
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NMR Nuclear Magnetic Resonance
NRP Nonribosomal Peptide
NRPS Nonribosomal Peptide Synthetase
NSF National Science Foundation
Ox Oxidation Domain
PBP Periplasmic Binding Protein
PCP Peptidyl Carrier Protein
PCR Polymerase Chain Reaction
PDB Protein Data Bank
PEG Polyethylene Glycol
PK Polyketide
PKS Polyketide Synthase
RMSD Root-Mean-Square Deviation
RND Resistance, Nodulation, and Cell Division Superfamily
RP Ribosomal Peptide
RPS Ribosomal Peptide Synthetase
RT Room Temperature
S-ribosylMet S-Ribosyl-L-Methionine
SA Salicylic Acid
SAD Single Wavelength Anomalous Dispersion
SIP Siderophore-Interacting Protein
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SMM S-Methyl-L-Methionine
TCEP Tris-2-carboxyethyl-phosphine
TE Thioesterase Domain
TFA Trifluoroacetic acid
TIM Triosephosphateisomerase
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
STRUCTURAL INSIGHT INTO MICROBIAL NATURAL PRODUCT PATHWAYS
By
Kunhua Li
May 2016
Chair: Steven D. Bruner
Major: Chemistry
Natural products include any substance produced by living organisms and
provide enormous structural diversities with unique biological relevant functions. Natural
products have been a surge in interest of new therapeutics and have been found
clinically effective for a variety of human ailments. The structures of the enzymes
involved in the natural product biosynthesis, delivery, utilization, degrading and repair
are critical to understanding the natural product pathways, thus facilitate the
development of relevant drugs. The identification and engineering of natural products
and their related pathways also facilitate the building, maintaining and developing of the
natural product library. My dissertation will focus on the natural product pathway related
proteins, including their functional and structural identifications and engineering.
The first three chapters provided an insight into the microbial siderophore
pathways, specifically the NRPS-based fuscachelin pathway in Thermobifida fusca. Two
critical proteins, FscJ and FscN involved in the fuscachelin delivery and utilization have
been functionally and structurally characterized. FscN is the first structurally
characterized siderophore interaction protein with associated functions; FscJ is the first
periplasmic binding protein corresponds for the mix-type siderophore delivery.
21
The next two chapters described ribosomal peptides and their biosynthetic
pathways. Chapter 4 detailed the microviridin biosynthesis in cyanobacteria. Microviridin
inhibits protease activity and is a potential antitumor drug lead. The structural
information of the two key enzymes in the microviridin J pathway, MdnC and MdnB
helped to understand the unique precursor peptide recognition. Chapter 5 described a
proteinous natural product, Y3 from fungus Coprinus comatus. Antiviral and antitumor
Y3 belongs to a novel lectin family.
The last chapter of my dissertation described an under-recognized aspect of
metabolite damage, which raises useless or toxic byproducts. This chapter provided an
example of MmuM facilitated (R,S)-AdoMet metabolite repair.
22
CHAPTER 1
MICROBIAL SIDEROPHORE-BASED IRON ACQUISITION AND MEDICINAL APPLICATION
1.1 Introduction of Microbial Siderophores
Iron is an essential element for all microorganisms and an important cofactor
required for many cellular, metabolic and biosynthetic processes1,2. The specific
acquisition of elemental iron is critical for a microbe’s survival and frequently
virulence3,4. Under common aerobic and neutral pH conditions, environmental ferric ion
has a low bioavailability concentration (10-9-10-18 M), which is usually below the
requirement for microbe optimum growth5. As a consequence, bacteria and fungi have
evolved diverse strategies to import and utilize iron under iron-deficient conditions
exemplified as6: the direct extracellular reduction of ferric compounds of low solubility to
soluble ferrous iron7; the acquisition of iron-bound heme or heme-containing proteins8;
the removal of metal from host iron-bound transferrin, lactoferrin or ferritin via specific
outer membrane receptors9; and the synthesis, secretion and utilization of iron specific
chelators, ferric-binding siderophores10.
Siderophores are low molecular weight compounds (400-1200 Da) with
extraordinary high affinity for ferric (less so for ferrous) ion, and tend to used negatively
charged oxygen as donor atoms2,10. Broadly, siderophores can be classified into three
categories depending upon the ligand moiety for Fe3+ coordination and include
catecholate/phenolate; hydroxamate/carboxylate and mixed-type, exampled as
enterobactin (1, Escherichia coli)11, aerobactin (2, E. coli)12 and mycobactin (3,
Mycobacterium tuberculosis)13 respectively. The different subclasses typically are multi-
dentate ligands, and show strong affinity for higher oxidation state of iron with
association constants of ~1030 or greater. Siderophores help maintain the intracellular
23
iron concentration between 10-7 and 10-5 M for microbes' survival and multiplication5. In
addition to iron, several siderophores also have the ability to seize and transport other
metals14.
Figure 1-1. Microbial siderophores with diverse scaffolds.
1.2 General Approaches of Siderophore Biosynthesis
Siderophores are commonly produced in the cytosol or peroxisomes4,15. Large
varieties of siderophores have been discovered in past decades with two different
pathways for structurally distinct siderophores biosynthesis. Most polypeptide-based
siderophores are biosynthesized via well-characterized, non-ribosomal peptide
synthetase (NRPS) systems. NRPSs are large multi-enzyme complexes responsible for
the synthesis of non-ribosomal biologically important peptides16. NRPS consists of
common domains in a gene cluster (or clusters17) as adenylation (A), condensation (C),
peptidyl carrier protein (PCP) and thioesterase (TE) along with other specific functional
domains includes epimerization (E), oxidation (Ox), methylation (Mt), and cyclization
24
(Cy). Additional accessary proteins may also present in the gene cluster to provide the
molecular building blocks for the NRPS machinery18,19. NRPS functional modules carry
out steps of monomer selection (A-PCP), modification (E, Ox, Mt); peptidyl chain
elongation (C), and cyclization/termination (Cy, TE). Siderophore NRPS assemblies
frequently initiate the peptidyl chain with a non-canonical amino acid building block
derived from an aryl acid, such as salicylic acid (SA) or 2,3-dihydroxybenzoic acid
(DHB) acting as the catecholate chelating moiety. The presence of thiazoline or
oxazoline rings in NRPS pathway based siderophores are also common, installed by
NRPS embedded Cy/Ox domains. Post-assembly line modification of siderophore
NPRs is less usual, a typical example of is the enterobactin C-glycosylation catalysed
by glycosyltransferase IroB in salmochelin (4) production20. In addition, a suite of marine
siderophores have a peptidic head group for ferric chelation, as well as fatty acid
tailoring that varies in length and saturation to tune the overall molecular
amphiphilicity21,22. Marinobactin (5) in is produced by Marinobacter sp. largely using an
NRPS machinery and further acylation with saturated and unsaturated C12-18 fatty
acids23,24. Comparability, amphi-enterobactin (6) in Vibrio harveyi BAA-1116 also
contains a fatty acid moiety. The amphi-enterobactin biosynthesis pathway contains a
bi-functional C-domain, which can accept amino acyl-phosphopantothenyl-PCP, as well
as acyl-CoAs as substrates25. Other recently described novel NRPS siderophore
scaffolds include mirubactin (7) from Actinosynnema mirum, which contains an
unprecedented hydroxamic acid ester26.
In contrast to NRPS systems, a number of non-polypeptide bacterial
siderophores are constructed by NRPS-independent synthetase (NIS) pathways27,28.
25
NIS siderophores include aerobactin, achromobactin (8, Pseudomonas syringae)29 and
desferrioxamine (9, Streptomyces griseus)30. These siderophores are usually
assembled from alternating dicarboxylic acid and diamine/amino alcohol building blocks
linked by amide or ester bonds. Recent examples of NIS-siderophores include
baumannoferrin (10) isolated from acinetobactin (the preliminary NRPS siderophore)-
deficient Acinetobacter baumannii AYE strain under iron-limiting condition31; and
putrebactins (11), the first identified unsaturated macrocyclic dihydroxamic acid
siderophores in Shewanella putrefaciens synthesized in a precursor-directed
mechanism32.
Figure 1-2. Typical siderophore pathway in Gram-negative bacteria. Figure was prepared with PyMol (https://www.pymol.org/) with PDB entries used listed: 3WDO, 2XMN, 4DK0, 4C48, 1FI1, 2W76, 2GSK, 2PFU, 5DH0, 2GZR, 4G1U and 4YHB.
26
1.3 Siderophore Secretion
Once synthesized, apo-siderophore will be secreted into the media to scavenge
iron4. Siderophore secretion is one of the least understood steps of high-affinity iron
acquisition in microorganisms. Several different secretion systems have been identified
as implicated in the process; comprise transporters from major facilitator superfamily
(MFS); and the efflux pumps of the resistance, nodulation, and cell division superfamily
(RND).
Many NRPS based siderophore gene clusters contain an MFS transporter-
encoding gene. MFS is one of the largest groups of transporters conserved from
bacteria to human for a wild spectrum of substrates including ions, carbohydrates,
lipids, peptides, nucleosides, and other primary and secondary metabolites33. The
majority of MFS members comprise 12-transmembrane α-helices (TMs), with some
containing 14-TMs or more33. Several MFS proteins from different subfamilies have
been structurally elucidated, but none of them associates with siderophore secretion34.
In Gram-negative E. coli, apo-enterobactin is transported by EntS, a 43-kDa archetype
MFS transporter encoded by the gene ybdA within the Fur-regulated ent-fep gene
cluster35. EntS exports enterobactin across the cytoplasmic membrane. Mutant ΔentS
(ΔybdA) shows significant reduction of enterobactin secretion with an increased release
of its byproducts. Comparably, in the Gram-positive B. subtilis, the MFS protein YmfE
was identified to participate in siderophore bacillibactin secretion during the single
mutants screening36,37. YmfE mutant strain has a severe bacillibactin secretion defect
and an eliminated bacteria growth in iron deficient medium.
27
In addition to the MFS class, the RND superfamily is an ancient and ubiquitous
group of proton antiporter widespread especially among Gram-negative bacteria
catalyses active efflux of heavy metals, drugs and siderophores38. The MexAB-OprM
system in Pseudomonas aeruginosa is the first RND identified in siderophore
(pyoverdin) secretion39. The mex operon is iron-regulated. In E. coli, as with MFS
transporters, TolC corresponds for the siderophore enterobactin efflux across the cell
outer membrane40,41. TolC is a trans-outer membrane protein and is an essential
component of RND pump AcrAB-TolC. AcrAB-TolC is a general transporter for bacteria
antibiotics and toxic compounds tolerance, and has been structurally characterized as a
whole42. The AcrAB-TolC pump consists with an AcrB:AcrA:TolC 3:6:3 ratio. AcrA
interacts with TolC through the hairpin domain and connect with AcrB through the β-
barrel and membrane-proximal domains. Whereas, the role of AcrAB in enterobactin
secretion is not entirely clear. Triple mutant ΔacrB/ΔacrD/ΔmdtABC, but not any
individual deletions, does result decreased enterobactin excretion43. AcrAD and
MdtABC are two other TolC-dependent RND pumps43. In contrast, the MmpS5-MmpL5
(or 4) RND efflux pump, a homolog of AcrAB has been identified as an critical
siderophore export system for the virulence M. tuberculosis44. MmpS5-MmpL5 is
regulated by the MarR-like transcriptional regulator Rv0678, whose open reading frame
is located downstream of the MmpS5-MmpL5 operon45. Mutant lacking the mmpS4 and
mmpS5 genes does not change the uptake of external carboxymycobactin (12), but
stops the cell grow under low iron conditions46. MmpS5-MmpL5 efflux system also
corresponds for the M. tuberculosis azole resistance47.
28
Besides the MFS and RND subclasses, IroC, a type-I ATP-binding cassette
(ABC) transporter similar to eukaryotic multidrug resistance (MDR) proteins in E. coli,
was previously suggested as responsible for the uptake of ferric-salmochelin48, is
proposed to be an active enterobactin/salmochelin exporter49. The double mutant
ΔentS/ΔiroC has a severely compromised growth rate, while IroC does not impact the
ferric-enterobactin/salmochelin utilization in the growth promotion49,50.
1.4 Holo-Siderophore Acquisition
Holo-siderophore acquisition is one of the key steps in iron assimilation. Gram-
negative bacteria possess an outer membrane layer and a peptidoglycan layer. Most
siderophores have molecular weights in excess of 600-Da, and their ability to permeate
the outer membrane is low10. Therefore, specific energy-dependent integral outer
membrane porin protein is essential to deliver iron-siderophore complex from the
extracellular into the periplasmic space, as typified by ferric-ferricrocin receptor FhuA51 ,
and ferric-enterobactin receptor FepA (and IroN)52. FhuA is a 22-stranded β-barrel that
spans the outer membrane, with an N-terminus plug domain accessible to the barrel
pore to facilitate ligand recognition and binding. FhuA also includes an N-terminus TonB
box, which forms a four-stranded β-sheet with TonB, a cytoplasmic membrane sigma
regulator53 with receptors specificity54. Meanwhile, ferric-citrate receptor FecA55 in E. coli
and ferric-pyochelin receptor FpvA in Pseudomonas aeruginosa56 belongs to the TonB-
transducer family, which possess a signaling domain upstream from the TonB box and
are able to self-regulate their own synthesis as well as their cognate siderophores.
TonB is part of the TonB/ExbB/ExbD energy transduction systems. The complex is
located in the cytoplasmic membrane, and provides the energy required for the active
29
transport of holo-siderophores through the outer receptor57. TonB undergoes energized
motion in the bacterial cell envelope58, interacts with ExbB4-ExbD2 complex59,60 to the
electrochemical gradient and initiate the energization61,62. The interactions promote iron
uptake through outer membrane transporters in a rotational mechanism.
The ABC transporter superfamily constitute a group of a transmembrane proteins
that perform ATP-coupled translocation of a wide range of substrates across cell
membranes63. ABC transporters deliver environmental ferric-siderophore to cytosol in
Gram-positive bacteria, or from periplasm to cytosol in Gram-negative bacteria. ABC
transporters have a common architecture that consists of a pair of transmembrane
domains (TMDs) embedded in the membrane lipidic bilayer, and a pair of nucleotide-
binding domains (NBDs) that are located in the cytoplasm63. Most siderophore ABC
transporters belong to type-II ABC importers, and their homolog, heme transporter
HmuUV in Yersinia pestis has been structurally characterized and identified to employ
an coupling mechanism distinct from that of other ABC transporters64. Some
siderophore-specific ABC transporters has been reported to be regulated by iron-
dependent regulators65,66. Siderophore ABC transporters interact with an external type-
III ABC-transporter periplasmic binding proteins (PBPs) to facilitate the substrates
delivery. Unlike type-I and type-II PBPs, which undergo significant domain transition
during substrate interaction, most siderophore type-III PBPs and have a relatively rigid
α-helix structure serving as the hinge between the two domains67 and ensures the
overall structural stability and also controls the domain movement between
‘open’/‘closed’ states upon specific substrate(s) recognition68,69,70. ABC transporter
meditated siderophore delivery in Gram-positive bacteria, as the YxeB-dependent
30
system in Bacillus cereus shuttle with an iron-exchange mechanism from ferric-
siderophore to apo-siderophore without iron reduction71,72.
In addition to ABC importers, the MFS transporter MirB in fungal pathogen
Aspergillus fumigates, a 14-TMs protein is reported to be responsible for the uptake of
hydroxamate siderophore N,N’,N’’-triacetylfusarinine-C (TAFC)73. No bacterial MFS has
been reported in holo-siderophore acquisition so far.
1.5 Siderophore Utilization
Imported ferric iron bound to siderophore must be released upon holo-
siderophore delivery to be made available to cellular machinery. One strategy for iron
release is through hydrolytic destruction of the holo-siderophore by esterases of the α,β-
hydrolase family of enzymes, represented by the E. coli fes iroE gene product, and is a
common strategy for macrolactone-based siderophores74,75. IroE is a periplasmic
trilactone hydrolase that tends to hydrolyse enterobactin at one ester to produce the
linearized trimers, which still display a considerable affinity to ferric ion. Thus, in addition
to iron assimilation, IroE is likely involved in the production of triscatecholate
siderophores74. On the contrast, periplasmic trilactone esterase Cee in Campylobacter
hydrolyses both apo- and holo- forms of enterobactin effectively, and further digests the
linearized trimer into dimers and monomers, which significantly lost the ferric affinity76.
Genomic evidence further demonstrates that Cee is involved in enterobactin mediated
iron uptaken76.
For cleavage-independent siderophore paths, iron release is commonly proposed
to be facilitated by single-electron reduction of the ferric-siderophore to the ferrous
oxidation state77. The relative weaker reduced ferrous-siderophore interaction allows
31
kinetic exchange with downstream iron-chelating sites found in cellular protein or small
molecules10. Two families of proteins have been identified so far involved in the
process, termed siderophore-interacting protein (SIP), and ferric-siderophore reductase
(FSR). FSR contains a unique C-terminal [2Fe-2S] cluster as C-C-x10-C-x2-C, and does
not show significant similarities to other known [2Fe-2S] proteins. FSR as E. coli FhuF is
a part of siderophore utilization system and has a sufficient redox potential to reduce
ferric-ferrioxamine78. FchR, a FhuF homolog in Bacillus halodurans DSM497 has been
reported to work in a three-component electron donor system, along with NADPH and
ferredoxin to reduce various iron chelates with optimal demonstrated reduction activity
against ferric-dicitrate79. Meanwhile, SIP is a flavoreductase. YqjH contains a covalently
bound FAD and reduce ferric-enterobactin with the presence of NADPH80,81. The yqjH
gene is regulated by YqjI, a Zn-dependent regulator82,83. YqjH homolog, FscN in T.
fusca has been structurally identified consists of a FAD-binding domain and an
NAD(P)H-interaction domain77. FscN contains a non-covalently bound FAD and reduce
ferric-fuscachelin-A (13) with NADH. Remarkably, the M. tuberculosis iron-regulated
ABC transporter, IrtA contains extended N-terminus FAD/NAD(P)H binding domains
similar to the SIP family84. As a result, IrtA has dual functions include iron delivery and
assimilation.
1.6 Siderophores as Therapeutical Drug Leads
Bacterial siderophores, themselves, have been shown to exhibit specific
antifungal or antibiotic activities85,86. Therapeutic iron chelators containing siderophore
scaffolds have also been used in the treatments of blood-transfusion requiring
diseases87. In addition, in the past decade, siderophore-antibiotic conjugates have been
32
developed as drug leads with reduced permeability-mediated drug resistance using
“Trojan Horse”-type mechanisms88. The strategy can impressively reduced permeability-
mediated drug resistance with target selectivity, and especically advantageous in MDR
pathogen control89.
Figure 1-3. Examples of siderophore related therapeutic drug leads.
Successful design of siderophore-drug conjugates contain, a siderophore moiety
that can be recognized and imported; a suitable linker stable enough in extracellular
environment but can be cleaved by enzymatic reactions either in cytoplasm or
periplasm; and an effective drug moiety, commonly of the β-lactam drug family90.
Siderophores-linked lactam antibiotics have an increased penetration through the outer
membrane91, which pathegen selectivity can be tuned with the conjugated siderophores.
Specifically, tris-catecholate siderophore-aminopenicillin conjugates (14) Inhibit Gram-
negative bacteria especially against Pseudomonas aeruginosa92; biscatecholate-
monohydroxamate siderophore-carbacephalosporin conjugates (15) are selective for
pathogenic Acinetobacter baumannii93. Another hexadentate siderophore-mediated
33
drug delivery system has been successfuly addressed with enterobactin backboned
siderophore-cargo conjugates in E. coli (or Pseudomonas aeruginosa)94,95.
Enterobactin-antibiotic conjugates, include β-lactam antibiotics ampicillin (Amp) and
amoxicillin (Amx) conjugates (Ent-Amp/Amx, 16) have 1000-fold decrease in minimum
inhibitory concentration (MIC) value against E. coli CFT073 relative to Amp/Amx
alone95. The enterobactin-mediated delivery is FepA dependent. The Ent-Amp
antibiotics selectivity can be further constricted with salmochelin modification (GlcEnt-
Amp/Amx, 17) to target specific pathogenic E. coli with iroN encoded salmochelin
receptor96. Salmochelin-modification also present a low mammalian cell toxicity96.
Lactam-family drugs can also be delivered with relatively smaller siderophores. The
bidentate siderophore-sulfactam BAL30072 (18) has a promising activity against multi-
resistant Gram-negative Bacilli97, include the strains express multiple β-lactamases as
meropenem-resistance Acinetobacter baumannii98. The antimicrobial efficacy of
BAL30072 can be further enhanced through the carbapenem combimation treatments99.
BAL30072 has been submitted to clinical trials phase-I in 2013100. Comparably,
siderophore-monocarbam conjugates MC-1 (19) possess in vitro and predicted in vivo
activity against MDR pathogen as Pseudomonas aeruginosa101,102. The MC-1/penicillin-
binding protein co-complex crystal structure established the molecular basis for the
recognition specificity and coupling activity103,104. Similar pyridone-monobactam
conjugate also possess the in vitro antibacterial activity against clinically relevant MDR
Gram-negative species105. MC-1 and its analogs are most likely to use the TonB-
dependent outer membrane siderophore receptor (PiuA, or PirA) as the primary mean
of entry105,106.
34
Besides siderophore-lactam conjugates, several lactam-independent
siderophore-drug candidates have been reported. The trihydroxamate siderophore-
fluoroquinolone conjugates (20) are sensitive to Gram-positive Staphylococcus aureus
SG511107. Lactivicin analog-phthalimide conjugates (21) use a wider set of TonB
receptors (compare with hydroxypyridone-lactams) to target penicillin-binding proteins
and accommodate the in vitro Gram-negative antibiotics activity108. Mycobactin-T
analog-artemisinin conjugate (22) displays high activity against extensively drug-
resistance (XDR) M. tuberculosis strains109,110. Core drug moisty artemisinin has an
antimalarial activity, but no antituberculosis activity.
1.7 Targeting Siderophore Pathways
Specific targeting of siderophore pathways is another applicable therapeutic
approach to address the microbial virulence. The disruption of iron recycling constrains
the microbial survival and reproduction4, and the interruption of holo-siderophore
acquisition is a potential therapeutically approach. Most siderophore-drug conjugates do
have a dividend effect on jamming the native siderophore acquisition. In addition,
reported antibiotic lasso-peptide Microcin-J25 (23) and its analogs target FepA111.
Lasso-peptide structurally hijack FepA to disrupt the ferric-siderophore acquisition and
reduce the microbial growth in vitro112. Several pathogen survive with mammals
siderophore through FepA113.
Seizing the siderophore biosynthesis to reduce virulence has received much
attention recently. In NRPS-based siderophore biosynthesis, acyl adenylate
intermediates, includes SA- and DHB-AMP analogs interrupt the substrate recognition
of the initial A-domain, subsequently inhibit a broad range of microorganisms with aryl-
35
capped siderophores production114. Sal-AMS (24) inhibit NRPS MbtA in M.
tuberculosis115,116, and significantly reduce the pathogen growth in mouse lungs in
vivo117. Other possible siderophore synthases target includes the specific NRPS-
accessary proteins. Biaryl nitrile (25) and its analogs target PvdQ, an essential N-
terminal nucleophile hydrolase in pyoverdine biosynthesis pathway, and decrease the in
vitro siderophore production, thus limit the growth of Pseudomonas aeruginosa under
iron-limiting conditions19.
Siderophore secretion is an alternative eligible pathogenic target. Pathogenic E.
coli requires siderophores for iron acquisition during infection, while exporters EntS and
IroC are important for the systemic virulence in a chicken infection model50. Outer
membrane transporter ΔtolC mutant E. coli also displays morphological defect in
minimal medium, and is probably caused by periplasmic enterobactin accumulation118.
Similar approaches have been successfully addressed in RND efflux system in M.
tuberculosis46. Exporter-deficient ΔmmpS4/S5 M. tuberculosis has an inhibited growth
rate, and the growth defect could not be rescued by external mycobactin supplement46.
The cytoplasmic mycobactin (and carboxymycobactin) accumulation provides a
possible self-poisoning inhibition mechanism. In this context, siderophore secretion is a
more advantageous target, as the biosynthesis or specific acquisition defect can be
overcome by using alternative ferric-carrying heme or siderophores46.
1.8 Conclusions and Insights
The chemistry and biology of siderophore-mediated iron acquisition is a complex
process and a key for step for microbial survival and virulency. Understanding the
siderophore-related pathways enables the design and development of siderophore-
36
scaffold drugs, as well as the therapeutic approaches in targeting the pathogenic iron
assimilation. Recent progress in siderophores and their related applications provide a
much-improved insight into the complex and important process of iron acquisition. The
developing clinical endeavors to target microorganism/pathogen in “iron battle” will
continue to stimulate research in the area.
37
CHAPTER 2
STRUCTURE AND MECHANISM OF THE SIDEROPHORE INTERACTING PROTEIN FROM THE FUSCACHELIN GENE CLUSTER OF Thermobifida fusca1
2.1 Siderophore Utilization Pathway in Thermobifida fusca
The specific acquisition of elemental iron is essential for a majority of
organisms119,120. Additionally, the cellular machinery to transport and assimilate iron
often contributes to the virulence of human microbial pathogens121. As described in the
previous chapter, various siderophore pathway-based therapeutical applications have
been developed during the past decades. These approaches majorly focus on
siderophore production and acquisition, while siderophore-based iron reduction is still a
blank area.
The intracellular reduction of ferric-siderophores is proposed to occur through
SIP or FSR family proteins. A preference of NADPH for reduction of bound flavin has
been demonstrated for SIP YqjH, along with the ability to reduce diverse iron chelates
including ferric enterobactin81. The ygjH gene is a part of the Fur regulon and under
transcriptional control of yqjI, a winged helix transcriptional regulator82,122. Interestingly,
the DNA-binding of YqjI is dependent on Ni2+ binding (not iron), and it is proposed that
the gene product helps regulate iron acquisition in conditions of high nickel ion
concentration, which can disrupt iron homeostasis83. FSR contains a unique C-terminal
C-C- x10-C-x2-C iron-sulfur cluster binding motif which does not show significant
similarities to other known [2Fe-2S] proteins78. FsR FchR from B. halodurans DSM497
has been reported to work in a three-component electron donor system, along with
1 Adapted with permission from Li, K., Chen, W.-H. & Bruner, S. D. Structure and mechanism of the
siderophore-interacting protein from the fuscachelin gene Cluster of Thermobifida fusca. Biochemistry 54,
3989-4000 (2015) DOI: 10.1021/acs.biochem.5b00354. Copyright © 2015 American Chemical Society
38
NADPH and ferredoxin to reduce various iron chelates with optimal demonstrated
reduction activity against ferric dicitrate123.
Figure 2-1. The fuscachelin siderophore pathway of T. fusca. A) Structure of the nonribosomal based peptide siderophore fuscachelin A along with, B) a 3-dimensional model of fuscachelin A-iron (green) complex. C) Schematic diagram showing the predicted roles of genes in the T. fusca siderophore gene cluster.
Our group have previously characterized a novel siderophore producing
biosynthetic gene cluster in the actinomycete T. fusca along with the structure of the
unique metabolite (Figure 2-1AB)124,125. The nonribosomal peptide (NRP) fuscachelin is
a mixed catecholate/hydroxamate with several unique structural features. The gene
cluster for fuscachelin contains many of the commonly found components for iron
acquisition (Figure 2-1C and Figure 2-2)124. The biosynthetic machinery includes NRP
synthetases (FscFGHI) and accessory biosynthetic enzymes FscABCDEK, in addition
to genes for transport and iron utilization. FscM is homologous to EntS-siderophore
transporters and is likely involved in export of the apo-siderophore. FscJ is a
membrane-anchored siderophore binding protein proposed to bind and deliver the ferri-
siderophore to the ABC transporter system. There are no genes for an ABC transporter
39
system specific to the fuscachelin gene cluster; however examination of the T. fusca
genome suggests a cluster (tfu_0336-0338) as a likely candidate with homology to the
E. coli FepCDG/FhuCB machinery126. These three genes are regulated by DtxR, an
iron-dependent transcriptional repressor that controls the expression of siderophore
gene clusters in T. fusca127. We are interested to exploit the thermostability inherit in T.
fusca to characterize the structure and mechanisms of the complex machinery involved
in siderophore biosynthesis and iron acquisition/utilization.
Figure 2-2. Annotated gene list of the T. fusca fuscachelin siderophore gene cluster. Genes with verified function or high homology to genes with predictable function are annotated as fscA-P.
Representatives of the two proposed families involved with intracellular iron
reduction are present in the T. fusca cluster. FscN is a flavoprotein of the SIP family and
FscP is a member of the FSR family shares high homology Fe/S cluster with E. coli
40
FhuF. It is uncommon to find representatives of both protein classes in a single
siderophore gene cluster. There is limited structural data available to provide a basis for
the function of the either superfamily in siderophore utilization. For the SIP family, there
is a preliminary X-ray diffraction analysis of YqjH from E. coli (but without structural
determination)80 and a structure of a homolog from S. putrefaciens has been solved and
deposited (PDB #: 2GPJ) as part of the Joint Center for Structural Genomics128. The 2.2
Å structure of the S. putrefaciens protein establishes the SIP class as a member of the
broad flavoreductase family with N-terminal β-barrel and C-terminal α/β/α sandwich
domains form the unique structural clades present in the SIP family across bacterial
species81. There are no structures available (or significant structural homologs) for the
FSR family of reductases. Here I report the structure and biochemical characterization
of FscN as a flavin-dependent iron reductase. The results provide a structural basis for
SIP function and insight into siderophore utilization in Gram-positive bacteria.
2.2 FscN is a Flavin Containing Siderophore Interaction Protein
2.2.1 FscN Contains a Non-covalently Bound FAD Cofactor
The fscN gene was cloned from T. fusca ATCC 27730 genomic DNA and the
protein was purified as a hexa-histidine fusion using standard chromatographic
techniques. The purified protein was bright yellow, indicative of a bound flavin-type
cofactor. The UV-visible spectrum of the isolated protein was consistent with flavin
adenine dinucleotide (FAD) in the oxidized form, with the absorption maxima at 388 and
459 nm and distinct shoulders located at 459 and 481 nm (Figure 2-3B). In order to
further identify the cofactor, the purified protein was denatured at high temperature and
the yellow supernatant was analyzed with LC-MS resulting in a molecular weight of
785.55 Da corresponding to the UV-active component, supporting FAD as the isolated
41
cofactor (Figure 2-3A). The protein precipitation formed during the denaturation
appeared white and was not UV active, suggesting the cofactor is bound to protein
through a non-covalently or labile covalent interaction. In order to confirm that the FAD
cofactor was redox-active in the context of FscN, the protein was reduced by sodium
dithionite under anaerobic conditions129. During the reaction, the 459 nm shoulder
decreased suggesting the disappearance of FAD over time, while the increase of the
600 nm shoulder peak indicates the formation of reduced FADH2 (Figure 2-3B).
Figure 2-3. Properties of the cofactor flavin bound FscN. A) LC-MS analysis of purified flavin cofactor, confirming the cofactor identification as non-covalent bond FAD. B) UV-Visible spectral of bound FAD (Ox) along with time points (16, 32, 48, 64, 80 min) of reduction with sodium dithionite, to FADH2 (+R, dotted line). The figure insert shows the full spectrum of reduced protein.
2.2.2 The Siderophore Utilization Protein FscN Binds Ferric-Fuscachelin
Isothermal titration calorimetry (ITC) supports FscN binding to ferric-fuscachelin
A with a reasonable binding affinity. FscN was titrated against ferric-fuscachelin-A and
42
an overall binding mode was modeled as an enthalpy driven process with calculated
binding affinity (dissociation constant) of 30 μM (Figure 2-4). We tested several other
ferric-siderophores for binding to FscN in order to determine the specificity of FscN.
Ferric-EDTA, ferric-citrate and ferric-2,3-dihydroxybenzoic acid (DHB) were all tested
using identical ITC conditions and no significant binding affinity was observed.
Figure 2-4. ITC analysis of ferric-fuscachelin A binding to FscN. The calculated binding constant and corresponding error is listed on the figure.
2.2.3 FscN Specifically Reduces Ferric Fuscachelin
Ferric reductase assays were performed anaerobically in a phosphate buffer to
further address the bioactivity of FscN81. Purified FscN showed no ferric reductase
activity toward ferric chloride, ferric-EDTA, ferric citrate, ferric 2,3-dihydroxybenzoic acid
43
or ferric enterobactin complex (data not shown). Relatively weak activity (40 nmol Fe2+
min-1 mg-1) toward purified ferric-fuscachelin A was detected with the presence of NADH
(Figure 2-5). The range of activity is consistent with that previously reported for YqjH,
which was reported to be 22.0 nmol Fe2+ min-1 mg-1 with a KM of 33 μM82. In contrast to
YqjH, inclusion of NADPH in the reaction in place of NADH did not result in any
observable reduction, suggesting that FscN functions as a NADH-dependent ferric
siderophore reductase.
Figure 2-5. FscN reduces ferric ion with the presence of NADH. The reaction was carried anaerobically with 1.0 mM NADH, 20 mM purified ferric fuscachelin A in 100 mM phosphate buffer, pH 7.5. Detection wavelengths of 340 nm were selected to monitor the consumption of NADH. NADH remained stable in the reaction system (red line) and converted into NAD+ with the presence of 100 µM FscN (Blue line). The reduction rate of FscN toward purified ferric fuscachelin A was calculated as 40 nmol Fe2+ min-1 mg-1.
2.2.4 Structure Determination with X-ray Crystallography
FscN protein crystals diffracted in the space group C2 and the structure was
solved using the deposited structure of the SIP from S. putrefaciens (PDB entry: 2GPJ)
as a molecular replacement search model. The final model was refined to 1.89 Å
resolution with two monomers of FscN present in the asymmetric unit (Figure 2-6, and
Table 2-1). The observed dimer is most likely a result of crystal packing and does not
represent the biological unit based on analysis of the interface using the PISA server130
44
and the size exclusion chromatography elution profiles are consistent with an FscN
monomer. The two monomers in the asymmetric unit are highly similar, with a total atom
RMSD 0.26 Å and the following discussion/figure representations will refer specifically
to the deposited monomer A.
Figure 2-6. X-ray crystal structure of FscN. A) The dimer present in the asymmetric unit shown in ribbon format with the flavin cofactor in licorice representation. B) One monomer of FscN illustrating the two N- and C-terminal subdomains and the location of an observed bound metal (shown as a Zn2+ ion).
The overall molecular structure of FscN consists of two subdomains: an N-
terminal FAD-binding domain and the C-terminal NADH-binding domain that are
connected by a linker region consisting of a two-strand β-sheet. The cofactor FAD is
clearly resolved in the electron density maps and non-covalently bound in the boundary
cleft between the two major domains. The FAD-binding domain has a six-stranded anti-
parallel β-sheet with a small α-helix connecting a long loop. The arrangement of the
NADH- binding domain forms a-β1-α-β2 architecture as is typical of the NAD(P)H
binding motif common to the classic Rossmann fold.
45
Table 2-1. X-ray crystallography statistics of FscN
Data Collection FscN complex with FAD
Wavelength (Å) 1.100
Resolution range (Å) 36.74 - 1.89 (1.96 - 1.89)*
Space group C 1 2 1
Unit cell (Å/º) 129.54 75.07 73.90 90 102.62 90
Total reflections 199130 (17743)
Unique reflections 54927 (4941)
Multiplicity 3.4 (3.3)
Completeness (%) 98.95 (94.18)
Mean I/sigma(I) 12.81 (2.64)
Wilson B-factor (Å2) 26.66
Rmerge 0.0587 (0.44)
Rmeas 0.0686
CC1/2 0.998 (0.899)
Refinement
Rwork / Rfree 0.189 / 0.219
Number of atoms 4643
protein 4141
ligand 140
water 362
Protein residues 523
RMS deviations
bond lengths (Å) 0.007
bond angles (º) 1.31
Ramachandran
favored (%) 99
outliers (%) 0
Clashscore 3.79
Average B-factors (Å2) 35.20
protein 35.30
ligand/ion 29.30
water 37.20
PDB entry 4YHB *Data values for the highest resolution bin are shown in parentheses;
46
Figure 2-7. Global multiple-protein sequence alignment of putative SIP proteins. Subgroups with differences in the N- and C-terminal regions are clustered. Conserved residues with green for NAD(P)H binding motif, yellow for FAD binding pocket and red for residues associated with putative metal binding. Abbreviations: Tfu, Thermobifida fusca; Nal, Nocardiopsis alba; Npo, Nocardiopsis potens; Sba, Shewanella baltica; Ptu, Pseudoalteromonas tunicata; Spu, Shewanella Putrefaciens; Eco, Escherichia coli; Cyo, Citrobacter youngae; Pfl, Pseudomonas fluorescens.
FscN displays significant structural homology to the diverse family of
FAD/NAD(P)H oxidoreductases. Using the structural homology program Dali131, several
NAD(P)H/ferridoxin reductases show Z-scores in the modest 17-19 range with overall
sequence identity between 10-15%. The highest overall similarities are with
flavohemoglobins, a three domain proteins that contain an iron-heme domain in addition
to the two ferridoxin-reductase subdomains common to FscN. The FAD cofactor is
bound in a typical conformation as seen with examples from this superfamily (Figure 2-7
and Figure 2-8). The negatively charged FAD diphosphate group is stabilized largely via
hydrogen binding interactions with His108 and Ser261. The isoalloxazine ring of flavin is
bound through aromatic stacking interactions between Tyr255, His53, Tyr89 and
47
His265. Additionally, an aromatic stacking arrangement holds the adenine moiety
between Trp256 and His108, and hydrogen bond interactions between the FAD and the
protein are seen with the 2’-hydroxyl of the ribose and the exocyclic amino of the
adenine to further reinforce the protein-cofactor interaction.
Figure 2-8. Two views of the flavin binding site illustrating key residues involved with cofactor interaction. A) Adenine view and, B) riboflavin view.
2.2.5 Proposed NADH Cofactor Binding Site in FscN
The presented structure is in a ‘closed’ conformation where the C-terminus is
occupying the likely NADH binding site. This is a common structural rearrangement
observed in this reductase class and the PDB database is represented by both
NAP(P)H bound and unbound states. In the ferridoxin reductase superfamily, two
electrons are commonly transferred from NAD(P)H to oxidized flavin followed by an
additional electron acceptor. In FscN, the NADH binding site is at the interface of the
two domains and can only bind while the protein is in an ‘open’ conformation.
Overlaying the structure of FscN with NADH-cytochrome b5 reductase132 (PDB entry
3W2G) allows insight into the predicted NADH binding to FscN. The open structure
NADH-cytochrome b5 reductase has the nicotinamide stacked with the flavin
48
isoalloxazine, in a position consistent with electron transfer. This stacking position in
FscN is occupied by Tyr255 at the C-terminus and disordering and/or displacing of the
terminus would need to accompany NADH binding. The structural arrangement has the
common features consistent with an established ping-pong, bi-bi mechanism of NADH
oxidation and substrate binding/reduction133. Residues observed to bind the NADH
cofactor in cytochrome b5 reductase are conserved in FscN, suggesting a similar
binding mode (see Figure 2-9A). In contrast to E. coli YqjH, with reported activity using
NADPH as cofactor, FscN has a longer loop between β12 and α3 to bind the adenine
part of the cofactor (see Figure 2-7). Structural homologs determined to have different
cofactor affinity also have slightly different sequence characteristics on this region.
Enzymes that utilize NADPH have a relatively shorter loop while NADH-dependent
proteins commonly have longer loop with notable presence of negatively charged
residues134,135. Previous research also suggests that negatively charged amino acids
(residue Glu173 in FscN) located on this loop interact with adenosine ribose moiety and
decrease NADPH affinity, enhancing the NADH utilization ability136. The structural
analysis along with the kinetics assays are consistent with FscN serving as a reductase
with cofactor specificity for NADH.
2.2.6 Metal Binding Site Adjacent to the Flavin Cofactor
From the calculated electron density maps, there is a pronounced metal site
adjacent to the hydrophobic side of flavin binding pocket (Figure 2-9B). The calculated
anomalous difference map shows a strong peak at a 15σ contour level, with a
wavelength of 1.100 Å. The electron density at the metal site is present up to 12σ
contour level in 2mFo-DFc map (Figure 2-9B), indicating the high occupancy of a heavy
49
atom. The metal ion is coordinated by three histidines, His53, His255, His256, with
nitrogen-metal distances of 2.18, 2.17 and 2.16 Å respectively. Three water molecules
also surround the metal center with the distances of 2.18, 2.18 and 2.27 Å giving a six-
coordinated metal center with ideal octahedral geometry. To identify the metal bound to
FscN, ICP-MS analysis of IMAC purified protein sample resulted in Ni2+ or Zn2+ (0.8 and
0.25 molar ratios respectively, Table 2-2). To exclude possible external metal ion
influence from the Ni2+-NTA resin used for protein purification, further analysis was
carried with the protein purified through ammonium sulfate precipitation. Analysis of this
sample resulted in an FscN: Ni2+: Zn2+ ratio 1: 0.22: 0.80, indicating a predominant Zn2+
form, however, the 0.22 molar ratio of Ni2+ is not insignificant, beyond that expected for
background.
Table 2-2. ICP-MS metal analysis of FscN. The protein was purified with Ni2+-NTA based immobilized metal ion affinity chromatography (IMAC) and ammonium sulfate precipitation (ASP) methods. Target protein FscN concentration was pre-adjusted to 100 ppb before the analysis.
Purification Method Mg Mn Co Fe‡ Ni Zn
IMAC Blank 1 0.957† 0.32 0.638 11.4 0.861 17.8
Sample 1 0.047 0.285 0.538 13.5 81.2 43.51
ASP Blank 2 0.622 -0.06 0.026 12.1 0.692 1.064
Sample 2 2.483 1.044 0.49 22.6 22.75 81.5 †Unit: parts per billion (ppb);
‡Fe analysis was carried out separately and has an inherently higher
background due to instrumental limit and error.
2.3 Structure of FscN Facilitate the Understanding of Siderophore Pathway
Presented is the first structure coupled with functional characterization of a SIP
flavoenzyme. The fate of iron-siderophores after import into cells is a complex and
important step of bacterial iron acquisition. The study of the overall process provides
basic biochemical insight into the complex molecular machinery and can provide details
50
into virulence mechanisms. The fuscachelin siderophore biosynthetic gene cluster
encoded in the genome of the moderate thermophile T. fusca is a useful model system
to study the overall mechanism. All common genes, including those for siderophore
peptide biosynthesis, export/import and utilization are present in the cluster, including
representatives implicated in intracellular iron reduction, the SIP, FscN and the FSR,
FscP. Iron is imported into T. fusca complexed with siderophore in the oxidized ferric
form and is at some point reduced to the ferrous state for uptake by cellular machinery.
There exist limited published biochemical characterizations of intercellular siderophore
iron reduction. As discussed, several pathways have been proposed, including
extracellular reduction and non-specific intracellular reduction paths. A majority of
bacterial siderophore gene clusters contain one representative of either the SIP or FSR
superfamily proteins. The FSR family is represented by E. coli FhuF, a ferredoxin
homolog containing an uncommon C-C-x10/11-C-x2-C binding motif, which shares limited
sequence similarity to other characterized 2Fe-2S ferridoxins. Examples from both
families have recently been reported to reduce iron-siderophores in vitro: FchR
reduction of ferric dicitrate123 and SIP YqjH reduction of ferric enterobactin81. It is not
clear if these functions are redundant or synergistic in siderophore pathways.
The fscN gene is part of the siderophore gene cluster of T. fusca. FscN is a
member of the SIP family and homologous to ViuB, required for siderophore utilization
in V. cholerae137. This family functions as NAD(P)H:flavin oxidoreductases, and
homologs are found to be widely distributed in bacteria. Two different subfamilies of
SIPs can be distinguished, primarily based on C-terminal sequence alterations138,139.
Group I (Figure 2-7), including FscN from T. fusca, contains an additional 15 - 20 amino
51
acids predicted to be a C-terminal α-helical elements with a conserved HH(K)x5DE
sequence. In contrast, group II, represented by E. coli YqjH, has a relatively shorter C-
terminus. All sequences have highly conserved Flavin cofactor binding motifs including
RxYT, DxV(F)xH, Gx2S and YWK(R) highlighted in yellow (Figure 2-7). As compared to
group II, group I SIPs have a more conserved NAD(P)H binding motif (TA-x3-EVL- x3-
GE) (shown in green). The NAD(P)H motif, QA-x3-SVL common to group II is less
common. Consistent with bioinformatics analyses, the group I YqjH uses NADPH
whereas in this report we demonstrate that the group II FscN utilizes NADH as a
reducing agent.
The structure of FscN contains a flavin cofactor bound in an overall conformation
typical of flavoreductases, with the isoalloxine ring in a planar configuration indicative of
oxidized FAD (Figure 2-8). The dinucleotide is in an extended orientation with the
terminal adenosine at the surface of the protein. The identity of the cofactor was
determined by protein denaturation followed by spectroscopic characterization of the
isolated cofactor. These, in combination with the presented structure, clearly suggest a
noncovalently bound flavin. This is contrast to the reported characterization of the E. coli
homolog, YqjH in which the cofactor was suggested to be covalently bound. Covalently
bound flavin is commonly linked through the C6 atom or/and the 8-methyl on the
isoalloxine ring through cysteine, histidine or tyrosine residues140,141. In our structure of
FscN, a well conserved His53 faces the 8-methyl in close proximity (3.8 Å), has no
obvious constraints to form a FAD adduct. We have shown that purified holo-FscN is
redox-active by the reduction of the enzyme with sodium dithionite, which produced
spectra consistent with the formation of FADH2. Efforts to show direct FscN reduction
52
with NADPH or NADH have been unsuccessful in our hands; possibly due to instability
of the reduced flavoenzyme in the absence of substrate. From analysis of the structure
and comparison with co-complex structures of flavoenzyme bound to NADH (highest
structural homology: NADH-cytochrome b5, PDB entry 3W2G, Figure 2-9), residues
likely involved with NADH binding are present in FscN, however, the very low overall
structural identity (17% to 3W2G), suggests a possible divergent function for FscN. We
have made considerable efforts to demonstrate the reduction of various iron chelates
with FscN, using the ferrine assay that was previously used to characterize the
reduction of YqjH and FchR82,123. Unlike FchR, we do not see reduction of ‘generic’ iron
chelates as ferric citrate, ferrichrome or ferric aerobactin. The postulated natural
substrate for FscN would be a ferric-fuscachelin A. Although we have access to
fuscachelin through isolation from T. fusca and through total synthesis, the generation
of sufficient quantities of pure holo-siderophore is a challenge. Regardless, we were
able to measure holo-fuscachelin A reduction activity of 40 nmol Fe2+ min-1 mg-1,
comparable to E. coli SIP YqjH with a reported activity 22.0 nmol Fe2+ min-1 mg-1.
Interestingly, YqjH is a NADPH dependent reductase while FscN only accepts NADH.
Commonly, the ratio of NAD+ to NADH inside the cell is high, while the ratio of NADP+ to
NADPH is kept low, therefore bacteria would use NADPH in vitro as a reducing agent
and NAD+ as oxidizing agent. The NADH dependence of FscN may suggest that the
siderophore utilization pathway in T. fusca has uncommon redox equilibrium.
Additionally, we have demonstrated a 30 μM FscN-ferric fuscachelin A binding affinity
and basic molecular docking calculations using Hexserver142 suggests that the
siderophore may bind the surface of the protein anchored by Glu62 and Asp264 to
53
correspond to the two positively charged D-Arg residues on the siderophore. The
pseudo-symmetric D-arginines in the natural product (see Figure 2-1B) are about 25 Å
apart, which is about the same distance as Glu62 and Asp264 on the protein suggesting
a possible binding mode.
Despite significant effort, we were not able to observe significant direct reduction
of flavo-FscN with NADH or NADPH nor were we successful at reducing a large variety
of ferric iron chelates with chemically (sodium dithionate) reduced FADH2-FscN.
Structurally, FscN is mostly similar to flavoheme proteins such as cytochrome b5
reductase which catalyze electron transfer from the two-electron carrier, NADH to the
one-electron carrier (heme) cytochrome b5. During iron-siderophore reduction, the
terminal electron acceptor (ferric-siderophore) has to be a single electron acceptor.
There is no equivalent heme binding protein near the FscN regulon, and based on the
requirement for a single electron reduction step, it is possible the 2Fe-2S of the FSR
FscP plays a role in facilitating single electron transfer from NADH to holo-siderophore.
There are multiple known reduction pathways that utilize NAD(P)H/flavin/FexSx as
component in electron transfer clusters, through in cis or in trans protein interactions.
Additionally, FscN shares 14% sequence identity and structural similarity (RMSD of 3.3
Å) with the 1,2-dioxygenase reductase from Acinetobacter sp. (PDB entry 1KRH)143.
Both enzymes have similar NAD(P)H and FAD binding domains, while the dioxygenase
reductase has an additional N terminal 2Fe-2S subdomain for substrate reduction.
Another example is the phthalate dioxygenase reductase (PDB entry 2PIA, RMSD of
2.8 Å) which utilizes FMN to mediate electron transfer from the two-electron donor
NADH to a [2Fe-2S] single-electron acceptor located on the C-terminus of the protein
54
144. In the case of T. fusca, electron transfer could proceed from NADH to flavin/FscN to
the 2Fe-2S/FscP (or vice versa) then to the iron-siderophore similar to the domain
arrangements of other members of flavoprotein reductase families (see Figure 2-
9)143,145,146,147,148. In this scenario, FscP would be in the position of the iron-porphyrin
heme ring as cytochrome b5 in cytochrome b5 reductase. Another possibility is that the
2Fe-2S/FscP acts as a holder of a single electron from the flavin semiquinone after a
ferric to ferrous-siderophore reduction. This can allow the ferrous-siderophore to
dissociate and a ferric-siderophore to bind followed by the turnover of the flavin
semiquinone to complete the stoichiometric reaction.
Figure 2-9. Domain arrangement of selected members of the flavoprotein reductase and electron transferase family. Listed proteins have structurally similar FAD (FMN)/NAD domains as compared with FscN, along with linked or dissociable electron carriers. The carrier can be either iron sulfur protein or a porphyrin binding domain. Abbreviations: FNR, ferredoxin-NADP reductase; b5R, cytochrome b5 reductase; MMO-C, methane monooxygenase; BenC, benzoate 1,2-dioxygenase reductase; NapR, NADH-ferredoxin NAP reductase; PDR, phthalate dioxygenase reductase; VanB, Demethylase oxido-reductase.
In the T. fusca gene cluster, FscN and FscP are separated by a single gene and
are part of the siderophore biosynthesis, transportation and utilization gene island. This
is a unique orientation as compared to most bacteria, where the siderophore gene
cluster contains one or the other SIP/FSR. For example in V. cholera, the vibriobactin
55
utilization protein ViuB (SIP) is located next to the nonribosomal peptide synthetase
VibF and a FSR-like 2Fe-2S protein is not identified in the organism149,150. In E. coli,
both SIP and FSR homologs can be found in the genome, both with characterized
function but neither is located within a single siderophore biosynthetic regulon.
Recently, DtxR has been identified as an iron-dependent transcriptional repressor that
regulates the expression of siderophore gene clusters in T. fusca127. It is possible that
the fuscachelin gene cluster is also regulated by the same gene under iron-deficient
conditions. Meanwhile, another iron-dependent transcriptional regulator in T. fusca Tf-
IdeR binds the toxPO sequence in the presence of a variety of divalent metal ions66.
Neither of these regulators, however has been shown to effect transcription of genes in
the fuscachelin siderophore cluster. We have conducted initial experiments to assess a
potential FscN/FscP, protein/protein interaction (Figure 2-10). FscN and FscP were co-
expressed in a pACYDuet-1 vector with N-terminus His-tag and C-terminus S-tag
respectively. Ni-NTA resin elution fraction from the co-expression was analyzed with
SDS-PAGE and His/S-tag Western blots. The positive S-tag Western blot indicates the
prospective protein-protein interaction between FscP and FscN supporting a possible
synergistic in vivo interaction. Unfortunately, significant efforts to overexpress and purify
FscP for in vitro analyses and/or structure determination have been unsuccessful in our
hands likely due to the significant instability of the holo-iron/sulfur protein.
An intriguing and unexpected aspect of our FscN structure is the observation of a
bound metal adjacent to the flavin site. It is not clear if this is an artifact of our
purification/crystallization or a natural aspect of the function of FscN. A bound zinc ion
that plays a structural role in FscN function is reasonable, but we did observe a
56
surprisingly high molar ratio of nickel ion bound to FscN, even in the absence of metal
affinity purification and a hexa-His-tag. Octahedral coordinate Zn2+ in rarely observed in
protein crystal structures151, whereas octahedral Ni2+ centers are more common. A
relationship exists between nickel ions and the E. coli SIP, YqjH. Transcription of the
yqjH gene is under control of the YqjI transcription factor, a nickel responsive
transcriptional repressor82. This system and others, demonstrate a correlation between
iron homeostasis and nickel concentration. There is no apparent yqjI homolog in T.
fusca and an interesting possibility is that nickel binding to FscN could play a role in the
regulation of enzyme activity. To date, the low in vitro reduction activity of FscN has
prevented us from testing this hypothesis. In addition, the three histidine residues are
not absolutely conserved in SIP members (See Figure 2-7) and suggest the observed
metal binding is likely restricted to a subset of Gram-positive bacteria.
Figure 2-10. S-Tag and His-tag Western blot for FscP-FscN coexpression. FscN and FscP were coexpressed in pACYDuet-1 with an N-terminus His-tag and C-terminus S-tag respectively. Ni-NTA resin elution fractions from coexpression experiments were analyzed with SDS-PAGE and His/S-tag Western blots. The positive S-tag blot (middle line) indicates the potential protein-protein interaction between FscP and FscN. Degradation of the 2Fe/2S FscP is evident in the SDS-PAGE gel.
57
The chemistry and biology of iron acquisition by small molecule siderophores is a
complex and fascinating process involving metal coordination, protein/protein
interactions and redox chemistry. The specific intercellular fate of iron-bound
siderophore is among the least understood steps at the molecular levels, there are
limited examples of direct biochemical demonstration of intracellular iron reduction and
transfer to cellular components. The presented structure and biochemical
characterization of T. fusca FscN provides insight into the role of the SIP family and
interplay with siderophores and possibly other protein players, such as FSR. In addition,
unexpected structural features and the inability to observe predicted in vitro function
suggests further investigation into the role of the SIP/FSR family.
2.4 Experimental Procedures
2.4.1 General Methods
Unless otherwise stated, reagents and chemicals were purchased from Fisher
Scientific or Sigma/Aldrich.
2.4.2 Cloning, Expression, and Purification of FscN
The gene for fscN was amplified with PCR from T. fusca (ATCC 27730) genomic
DNA with the primers: FscN_Nterm (5’-GCG GGA TCC ATC ACC GCA ACC GTG), and
FscN_Cterm (5’-GCG AAG CTT CTA CTC GTC GTC ATC GTC). The PCR products
were purified through agarose gel-electrophoresis and cleaved with the corresponding
restriction endonucleases. The resultant fscN gene was then ligated into the expression
vector pET28a and transformed into E. coli BL21(DE3)pLysS cells for expression.
Cultures (1L) were grown to OD600 0.6 - 0.8 at 37°C, and overexpression was initiated
by the addition of 100 μM IPTG and growth continued for 16 hours at 16°C before
harvesting by centrifugation. Cell pellets were resuspended in 25 mL 20 mM Tris-HCl
58
pH 7.5, 0.5 M NaCl and lysed at 14,000 psi through a nitrogen-pressure microfluidizer
cell (M-110L Pneumatic). The lysate was clarified by centrifuged at 10,000 g for 40 min
at 4°C. FscN was purified using metal affinity chromatography (HisPur Ni-NTA Resin,
Thermo Scientific). After binding for 1 h, the resin was washed with 4 × 10 mL of 20 mM
Tris-HCl pH 7.5, 0.5 M NaCl, 25 mM imidazole, and the bound protein was eluted with 3
× 4 mL of wash buffer with 250 mM imidazole. The resulting yellow elution solution was
dialyzed against 1 L 20 mM Tris-HCl pH 7.5, 100 mM NaCl for 16 h and further purified
with HiTrapQ HP anion exchange column (column size 5 mL, GE Healthcare, AKTA
FPLC System) and HiLoad 16/60 SuperDex-200 gel filtration chromatography (GE
Healthcare). To rule out contaminating Ni2+ from Ni2+-NTA based purification, an
alternative protein purification strategy using ammonium sulfate precipitation (ASP) was
also used for ICP-MS analysis. Briefly, the cell pellet was resuspended and lysed as
described above and ammonium sulfate was added into the supernatant and the
concentration was increased stepwise, from 10%, 30%, 50%, to 80% saturation. For
each step of addition, ammonium sulfate was gently added to the tube over 5 min and
rocked for another 5 min to facilitate dissolution. When all of the ammonium sulfate was
dissolved, the tube was placed on ice for 30 min to allow further precipitation of protein
before harvesting the precipitant through centrifugation (3,000 g for 10 min, 4°C). The
precipitant was resuspended in 5 mL 20 mM Tris-HCl pH 7.5 and analyzed at each
stage, and the target protein was recovered in the 50% precipitant fraction. The protein
was dialyzed and further purified by anion-exchange and gel filtration chromatography
as described above for Ni-NTA purified protein.
59
2.4.3 FAD Determination, Reduction and Enzyme Kinetics
In order to determine the cofactor composition, purified FscN was denatured by
heating at 95°C for 20 min. After removing the precipitate by centrifugation, the
supernatant was analyzed by LC-MS (Agilent 6130, SB-C18 column) with a gradient of
2 - 98% CH3CN in aqueous 0.1% TFA over 17 min at a flow rate of 1 mL min-1.
Preparative HPLC (GraceVydac-C18) purification was carried out with a linear gradient
of 3 - 40% methanol in aqueous 0.1% TFA over 30 min at a flow rate of 10 mL min-1.
Detection wavelengths of 320/460 nm were chosen to monitor the elutions. The m/z 784
peak in LC-MS represent FAD as cofactor.
Reduction of the FAD cofactor in vitro was assayed as described previous80.
FscN at a concentration of 1.5 mg mL-1 in a 1 cm cuvette was reduced by addition of an
excess of sodium hydrosulfite, and the spectrum was subsequently recorded at 16 min
intervals (16, 32, 48, 64, and 80 min). The detection wavelength of 459 nm was
selected to determine FAD reduction.
The FscN catalyzed reduction assay was performed anaerobically in 100 mM
potassium phosphate buffer, pH 7.5 at 37°C. For substrates ferric-EDTA, ferric chloride,
ferric citrate, ferricyanide and ferric 2,3-dihydroxybenzoic acid, the enzyme
concentration was set at 10 μM with the NADH concentration from 0.5 - 2.0 mM. 0.6
mM of the ferrous indicator 3-(2-pyridyl)-5,6-bis(2-furyl)-1,2,4-triazine (ferene) was
added to monitor the formation of ferrous ion by measuring the absorbance at 595 nm.
For substrates ferric enterobactin, and ferric fuscachelin A, due to the self absorbance
change of the Fe2+/3+-siderophore complex near 600 nm, reactions were monitored
through the concentration change of reactant NADH with the detection wavelengths of
60
340 nm. Final concentration of enzyme, NADH and substrate were set at 10 μM, 0.1
mM and 0.2 mM respectively and the reaction rate was calculated using ε340(NAHD)=
6220 M-1 cm-1.
2.4.4 Purification of the Ferric-Fuscachelin Complex
Apo fuscachelins A-C were produced from T. fusca culture as previously reported
with modifications and identified correspondingly152. Briefly, T. fusca spores (ATCC
27730) were cultured in 5 mL LB broth at 55°C and 150 rpm shaking overnight then
cells were fully exchanged into 5 mL of iron-deficient Hägerdal medium before
inoculating 1 L iron-deficient Hägerdal medium and growth continued for 7 d at 55°C.
Cell pellets were collected by centrifugation and extracted with methanol. The methanol
extracts were concentrated and subjected to preparative HPLC by using a Vydac
218TP1022 protein and peptide C18 column (250×22 mm, 10 μm). A linear gradient of
2-50% methanol in 0.1% TFA and water was run over 30 min at 10 mL min-1 and
fractions collected at 17.6, 18.2 and 19.1 min were confirmed to be fuscachelin C, B,
and A, respectively. Purified fuscachelin A was incubated with ferric ammonium sulfate
in a 1:10 ratio and the resulting ferric fuscachelin A complex was purified using
preparative HPLC with a linear gradient of 3 - 80% methanol in 5 mM NH4OAc over 25
min at 10 mL min-1. Apo-fuscachelin A with an elution time 11.2 min was converted
quantitatively into holo-fuscachelin A with an elution time 14.6 min.
2.4.5 Purification Ferric-Enterobactin
For enterobactin production and purification, we applied a novel approach to
produce enterobactin by overexpressing the putative enterobactin exporter EntS gene in
E. coli. The entS gene was cloned from E. coli BL21(DE3) and ligated into pET16b for
overexpression. The EntS/pET16b plasmid was transformed into E. coli BL21(DE3) and
61
cell growth was performed in iron-deficient M9 media. Supernatant from 6 L of cultured
E. coli (grown to stationary phase) was lyophilized to 150 mL and extracted with equal
volumes of ethyl acetate four times and concentrated. The residue was dissolved in
methanol for further LC-MS analysis and HPLC purification153. LC-MS analysis was run
in a linear gradient of 2-98% acetonitrile in 0.1% trifluoroacetic acid /water over 15 min
at 0.2 mL min-1. Fractions eluted at 11.86, 12.91, 13.81 and 13.97 mins have molecular
weight of 464.2, 687.3 669.2 and 669.2 Da. Combined with UV-vis spretra, we assigned
the four fractions to be (DHB-Ser)2, (DHB-Ser)3, apo-enterobaction and holo-
enterobactin respectively. The total methanol extract was mixed with 1 mM ferric
ammonium sulfate before preparative HPLC purification. A linear gradient of 10-50%
acetonitrile in 0.1% trifluoroacetic acid and water was run over 25 min at 8 mL min-1 and
50-98% acetonitrile was carried sequentially from 25 to 30 min. Fractions collected at
26.0 min were confirmed to be holo-Fe+3-enterobactin by mass spec and UV-Vis
analyses.
2.4.6 Metal Analysis
The hexa-histidine tags of purified FscN from both nickel NTA affinity
chromatography and ammonia sulfate precipitation were removed using a standard
thrombin cleavage protocol. Briefly, 1 μL of thrombin (1 U L-1, Novagen) was mixed with
10 mg of protein in 1 mL thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5
mM CaCl2, pH 8.4) and incubated for 16 hours at room temperature. Cleaved protein
was further purified using size exclusive chromatography (as described above). FscN
containing fractions (8 mL) were dialyzed against 1 L metal-free 0.1 M ammonium
acetate pH 6.5 for 16 h (pretreated with 5% w/v Chelex-100 Resin for 4 h) and diluted
62
with the same buffer to 10 mL (final concentration of 0.3 mg mL-1). Concentrated nitric
acid (trace metal grade, Sigma) was then added to a final concentration of 1%. A blank
10 mL reference was also prepared in parallel using identical procedures. Samples
were analyzed with inductively coupled plasma mass spectrometry (ICP-MS) at the
Center for Applied Isotope Studies, University of Georgia. Cu, Zn, Mn, Mg, Ni, Co and
Fe were analyzed for each sample along with the blank reference.
2.4.7 Isothermal Titration Calorimetry Binding Determination
FscN-siderophore binding affinities were determined using a MicroCal iTC200
isothermal titration calorimeter system. FscN was purified with Ni-NTA based
immobilized metal ion affinity chromatography followed by size exclusive
chromatography. Ferric-fuscachelin A was prepared from the isolated natural product
and purified to homogeneity154. 200 μL of 100 μM FscN in 10 mM HEPES-NaOH, 50
mM NaCl, pH 7.5 was placed in the ITC reaction cell and 40 μL Fe+3-fuscachelin A (1
mM) was titrated into the protein solution over time at 25°C. Heat change during the
reaction was detected and recorded for binding affinity calculation. All titrations were
repeated in triplicate. The binding parameters and corresponding error are calculated
using the Origin software package155.
2.4.8 Coexpression of FscN-FscP and Western Blot Analysis
FscN-FscP coexpression was carried out using the pACYDuet-1 vector. The fscN
gene was ligated into the multiple cloning site 1 (MCS1) with an N-terminal hexa-
histidine tag and the fscP gene was cloned from genomic DNA (primers FscP_Duet_N
and FscP_w/oSDuet_C, Table 2-3) and inserted into the MCS2 site with a C-terminal S-
tag. Cultures were grown to OD600 0.6 at 37°C and induced at 16°C overnight with 250
μM IPTG. Proteins were purified with IMAC as described previous and the elution
63
fraction was separated by SDS-PAGE. His-tag Western blot and S-tag assays were
carried out by using standard techniques. The proteins were transferred onto
polyvinylidene difluoride (PVDF) membranes (Bio-Red) for His-tag Western blot using a
poly-histidine monoclonal antibody (Sigma) as the primary antibody and goat anti-rabbit
IgG-HRP as secondary antibody. Western blot signal was developed with SuperSignal
West Pico Chemiluminescent Substrate (Thermo Forma) and detected on KODAK RP
X-OMAT film. Nitrocellulose membrane (S∙Tag AP Western Blot Kit, Novagen) was
used in S-tag Western blot. The transferred membrane was incubated in TBST + 1%
gelatin (TBST: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20) for 15 min to
block excess protein binding sites. Membrane was further incubated in 1/5,000 S-
Protein alkaline phosphatase conjugate TBST dilution for 15 min at room temperature
and developed with NBT + BCIP in alkaline phosphatase buffer. All Western blot
experiments were repeated twice to confirm the results.
Table 2-3. PCR primers used for FscN related cloning
Primer Vector Sequence
FscN_N pET28a/pACYDuet GCGGGATCCATCACCGCAACCGTGACG
FscN_C pET28a/pACYDuet GCGAAGCTTCTACTCGTCGTCATCGTC
FscP_Duet_N pACYDuet GCGCATATGATGACCCGTCAGTGTC
FscP_w/oSDuet_C pACYDuet GCGGATATCGTTGCGCAGCCACTC
C-EntS-Nhe1 pET16b GCGGCTAGCACTGTCGGACGCTGT
N-EntS-NcoI pET16b GCGCCATGGATAAACAATCCTGGC
2.4.9 Crystallography, Structure Determination, and Refinement
Initial crystal screening for FscN was performed in vapor diffusion sitting drop
format with commercial matrix screens (Hampton Research). Optimization of initial
conditions gave rod-shaped crystals with approximate dimensions of 400×100 ×100 µm
64
using a reservoir solution 0.95 M ammonium sulfate, 0.1 M Tris-HCl pH 6.6 and 35% v/v
2-methyl-2,4-pentanediol (hanging drop format, room temperature). Typically, crystals
appeared in 4 weeks and grew to maximum dimensions in 8-10 weeks. Crystals
suitable in size were harvested and frozen in liquid nitrogen using additional 30%
glycerol as a cryoprotectant. Diffraction data were collected at the PXRR X25 beamline
at Brookhaven National Labs National Synchrotron Light Source and processed with
XDS156 to a resolution of 1.89 Å in space group C2 (Table 2-1). The asymmetric unit
contains two molecules of FscN. Deposited SIP protein from S. putrefaciens (PDB code
2GPJ, 32% sequence identity) was used as starting models for molecular replacement
in PHASER157. Refinement was performed in PHENIX.REFINE158 in PHENIX 1.7.1159,
and model building was performed using COOT160. Based on the refined model, the
sigma-weighted, simulated composite omit maps and anomalous difference electron
density maps were calculated with PHENIX.MAPS159. Graphical representations were
prepared with PyMOL161. Several independent PCR and sequencing confirmed that the
construct of fscN from T. fusca ATCC 27730 deviates from the published genome of T.
fusca strain YX at one codon: position 99, is an Arg (AGA) in ATCC 27730 while a Gly
(GGA) in T. fusca strain YX. Arg99 is located on the surface of the resolved structure of
FscN and is outside the active site.
65
CHAPTER 3
STRUCTURE AND FUNCTIONAL ANALYSIS OF THE SIDEROPHORE PERIPLASMIC BINDING PROTEIN FROM THE FUSCACHELIN GENE CLUSTER OF
Thermobifida fusca1
3.1 Periplasmic Binding Protein Facilitates Siderophore Delivery
As noted in the chapter 1, iron is an essential cofactor required for the majority of
organisms, functioning as a global regulator for many cellular, metabolic, and
biosynthetic processes2,10. Importantly, siderophore based iron acquisition is commonly
critical for microbes’ survival and virulence1. Holo-siderophore uptake across the cellular
membrane(s) is similar in both Gram-positive and Gram-negative bacteria (see Figure
1-1). For Gram-positive bacteria, the FepBCDG-like multifunctional ABC-transporter
systems acquire and deliver the holo-siderophore through a siderophore-shuttle iron-
exchange mechanism71,72 with the assistance of a FepB-like periplasmic binding protein
(PBP)162.
Most siderophore PBP are type III PBPs67. Unlike type I and II PBPs, which
undergo significant domain transition during substrate interaction, type III PBPs have a
relatively rigid α-helix hinge between the two domains to controls the domain movement
between ‘open’/‘closed’ states67. Type III PBPs accept a wide range of substrates,
including free ion, ferric-siderophores, or ferric-hemes162. Aside from its natively
produced ligand, a particular PBP can also accept alternative ligands in vivo. The E. coli
PBP FhuD recognizes gallichrome as well as other structurally similar hydroxamate-
type siderophores, including the antibiotic albomycin163,164. To date, several type III
1 Adapted with permission (License Number 3779401197741) from Li, K. & Bruner, S. D. Structure and
functional analysis of the siderophore periplasmic binding protein from the fuscachelin gene cluster of Thermobifida fusca. Proteins Struct. Funct. Bioinforma. 84, 118-128 (2016). DOI: 10.1002/prot.24959. Copyright © 2015 Wiley Periodicals, Inc.
66
siderophore PBP structures have been solved through X-ray crystallography, illustrating
‘open’ and ‘closed’ conformations relevant to the substrate binding mechanism. For
example, ligand free FitE from E. coli has been identified in two different conformations
from the same crystallization condition and comparison of these two states shows a
change in the size of its binding pocket165. Likewise, the S. aureus siderophore receptor
HtsA undergoes a localized conformational shift upon staphyloferrin recognition166.
Additionally, recent NMR studies have provided insights into the structural dynamics of
the E. coli FepB in the solution phase68. Although most PBPs share a similar function
and have a relatively conserved general domain arrangement, the sequence diversity
within the superfamily is high and most solved PBP structures have a significant
different structural fold compare with each other, which provide difficulties to establish a
unified PBPs siderophore interaction mechanism. Moreover, most reported PBPs only
accept single type (hydroxamate or catecholate) siderophore. There is little information
about the mixed-type siderophore-PBP interaction.
Here I present the structure of FscJ, a type III periplasmic binding protein and its
interaction with a mixed-type siderophore, ferric-fuscachelin from T. fusca. The
siderophore fuscachelin is a secondary metabolite produced by a nonribosomal peptide
synthetase (NRPS) gene124,152 and is a catecholate /hydroxamate type siderophore with
positively charged D-Arginine subunits. The fscJ gene locates in the fuscachelin
biosynthetic gene cluster and encodes for the siderophore PBP, predicted to deliver the
ferric-fuscachelin A to the ABC transporter system137. To gain insight into the complex
recognition and transportation mechanisms of the unique siderophore fuscachelin, we
overexpressed FscJ and determined its structure through X-ray crystallography. Several
67
unique structures detail ligand-free conformational changes at different pH values,
indicating complex interdomain flexibility of the siderophore receptors. Additionally,
measured binding affinity of ligands and molecular docking provide valuable insight into
FscJ siderophore recognition mechanisms.
3.2 Characterization of FscJ as a Siderophore Binding Protein
3.2.1 FscJ and Fuscachelin Delivery
FscJ is a representative of the broad family of lipid-anchored periplasmic binding
proteins, key players in siderophore-mediated iron acquisition. The overall process of
siderophore-mediated iron acquisition is complex and most of the common protein
players are present in the T. fusca fuscachelin gene cluster. There is no gene encodes
for the ABC transporter in fuscachelin gene island. However, the cluster tfu_0336-0338
in T. fusca genome is a likely candidate with a high homology to the E. coli
FepCDG/FhuCB machinery126. As common to the PBP family, FscJ has a predicted N-
terminal transmembrane α-helix (residues 46-64)167. Our expression of full length FscJ
resulted in insoluble protein that was incorporated on E. coli membrane (Figure 3-1). In
order to improve the heterogeneous expression level and produce soluble protein, the
fscJ gene was engineered to exclude the transmembrane and lipidation sites (1-78).
Eight additional N-terminal residues (79-86) were also omitted due to predicted
disorder139. Purified His6-FscJ has a molecular weight of 35.6-kDa as measured in
MALDI-TOF mass spectroscopy, but appears as a dimer form on SDS-PAGE and in
size-exclusive chromatography (Figure 3-1).
68
Table 3-1. FscJ crystallization data collection and processing
Crystals FscJ_SeMet FscJ_P41 FscJ_ P21 FscJ_I222
Wavelength (Å) 0.9786 0.9786 0.9786 0.9786
Space group I222 P41 P21 I222
a, b, c (Å) 102.5 107.0 136.0
73.0 73.0 135.3 64.41 135.6 80.9
106.6 107.1 136.3
α, β, γ (°) 90 90 90 90 90 90 90 91.98 90 90 90 90
Mosaicity (°) 0.32 0.18 0.19 0.18
Resolution (Å) 41.76-3.20 (3.31-3.20)*
38.36-2.44 (2.53-2.44)
46.53-2.84 (2.94-2.84)
37.78-2.59 (2.68-2.59)
Total reflections 99379 (9626) 162648 (15443) 166896 (15936) 184378 (18691)
Unique reflections 12647 (1223) 26090 (2528) 32606 (3174) 24621 (2441)
Completeness (%)
99.7 (99.0) 99.6 (97.2) 99.7 (98.0) 99.9 (99.8)
Multiplicity 7.9 (7.9) 6.2 (6.1) 5.1 (5.0) 7.5 (7.7)
<I/σ(I)> 18.35 (3.09) 13.5 (3.3) 13.7 (2.2) 25.2 (3.8)
Wilson B factor (Å2)
79.7 17.0 12.6 33.4
Rmerge (%) 0.114 (0.92) 0.107 (0.82) 0.147 (0.78) 0.049 (0.57)
Rmeas (%) 0.126 0.118 0.166 0.053
CC1/2 0.998 (0.895) 0.997 (0.939) 0.992 (0.793) 1.000 (0.995)
Rwork (%) 0.173 (0.230) 0.1823 (0.267) 0.230 (0.282)
Rfree (%) 0.2267 (0.330) 0.247 (0.319) 0.263 (0.306)
No. of atoms 4411 8547 4282
protein 4325 8524 4247
water 86 23 35
Protein residues 556 1103 548
RMS deviations
bond lengths (Å) 0.008 0.012 0.004
bond angles (º) 1.01 1.39 0.74
Ramachandran
favored (%) 97 93 96
outliers (%) 0 0.64 0.19
Clashscore 1.78 4.16 2.66
Average B-factors 45.80 40.10 77.00
protein (Å2) 45.90 40.10 77.10
water (Å2) 42.20 36.20 66.10
PDB entry 5DH0 5DH1 5GH2 *Values for the outer shell are given in parentheses
69
Figure 3-1. FscJ purification and characterization. A) Size exclusive column purification of FscJ (with hexa-Histag). FscJ elution time is similar to proteins with molecular weight ~ 60-kDa; B) SDS-PAGE of purified FscJ with/without hexa-Histag (left/right line) with an observed molecular weight between 43~56-kDa; C) MALDI-TOF-MS analysis of FscJ (with hexa-Histag) provide a single mass of 35.6 kDa and, D) SDS-PAGE and Western Blotting analysis of full-length FscJ.
3.2.2 FscJ Structure Determination with X-ray Crystallography
FscJ structure was determined with SAD phasing. MR-SAD greatly facilitated the
final atomic coordination determination. Native FscJ protein crystals diffracted in
multiple space groups I222, P21 and P41 with a resolution range between 2.4-2.8 Å
(Table 3-1). The I222 crystal form was obtained exclusively at pH 4.6 condition, P21
crystals in pH 5.6 condition while P41 crystals were harvested in either pH 6.5 or 7.5
condition. The overall protein has a conserved two-domain structure (Figure 3-1A). The
N-terminal domain of FscJ monomer has a three-stranded parallel β-sheet sandwiched
by α-helices α4 and α5. Meanwhile, the C-terminal domain contains a five-stranded β-
sheet surrounded by α-helices on the protein surface. The two domains are connected
with a 40 Å long α6 spanning the whole molecule. A large cleft is formed between two
domains with an interface surface of 750 Å2 (PISA server)130.
70
Figure 3-2. Overall structure of FscJ. A) FscJ dimer (two orientations) present in the asymmetric unit contains two domains (N-terminal in blue and C-terminal in yellow) connected by an α-helical hinge (green). One monomer is shown in ribbon format and the other surface representation. A fragment of the hexa-Histag fragment is shown at the interface of one monomer. B) FscJ forms two different overall orientations when crystallized in different pH value conditions, F1 (I222) form in green and F2 (P41) in grey; C) Cα-Cα difference map between F1 and F2 forms (calculated with Matlab), the absolute value of the difference between the two matrices is displayed. The arrows indicate the center of motion.
FscJ forms a dimer in crystallographic asymmetric unit. The surface of
interdomain cleft is positioned close to the neighbouring α’6 hinge. A fragment of the
hexa-Histag is present between the interfaces of protein molecules contributing to the
71
dimerization (Figure 3-2A). Hydrogen bonds between Gln217 and His61 (of the hexa-
Histag), Glu221 and His61, His61 and neighbouring Glu’249, largely stabilized the
intermolecular interactions. The two monomers in both I222 and P41 space groups are
very similar, with a total atom root-mean-square deviation value (RMSD) of 0.18 Å
(I222) and 0.26 Å (P41) respectively. Meanwhile, the RMSD between monomers in I222
and P41 crystal form is 0.86 Å. Interestingly, space group P21 form crystal appears in a
intermediate state, with two out of four monomers similar to I222 form, and the other two
similar to P41 form. The following discussion/figure representations will focus on
monomer A in the I222 and P41 structures and refer them as Form 1 (F1) and Form 2
(F2) respectively.
Figure 3-3. Siderophore binding pockets in the SBP family (solvent accessible surface representations. A) The FscJ binding pocket spanning the cleft between the N- and C-domains. The conserved Arg164 on β2α4 is shown in blue, along with negative charged amino acids proposed to interact with fuscachelin in red; B) structure of fuscachelin A; C) YfiY surface with siderophore schizokinen in the binding pocket; D) SirA with staphyloferrin B.
The FscJ siderophore binding pocket is located on the top of central cleft
between the N-domain α2 and C-domain β7α11 loop region (Figure 3-2A). Residues
72
lining the pocket preliminary contain hydrophobic side chains; with one basic residue
(Arg164) locate in the pocket center, and three acidic residues (Glu279, Glu302 and
Asp310) at the side (Figure 3-3A). Like most of the other class III/α-helix bridged SBPs,
the N- and C-domains of FscJ overlay well in the F1 and F2 structures, while the size of
the binding pocket is altered with the protein C-domain movement. FscJ structural
changes between F1 and F2 were calculated quantitatively using a Cα-Cα (backbone
alpha carbon) difference distance map (Figure 3-2C). Between F1 and F2, there is no
observable Cα movement within N-domain (91-206) or C-domain (236-361) alone.
Meanwhile, the distance between N-domain Cα and C-domain Cα move up to 3.4 Å.
From the Cα-Cα difference distance map, FscJ domain movement is centered at the
residue region 222-228, and consists well with DynDom server calculation168. The
relatively ‘closed’ form, F1, has a hinged motion rotation angle of 5.5° compare with the
‘open’ form F2 (Figure 3-2B). F1 loop region between C-domain β4β5 shifted 3 Å
towards N-domain. There is also a 2 Å shift of F1 Phe229 on α9β6 loop towards the
middle substrate-binding cleft. The minimum distance between N-domain β2α4 loop
Arg164 and Phe281 in F1 and F2 structure shifted 3 Å closer to the middle binding cleft.
Trp258 on C-domain β5α8 loop also moves 3 Å closer to Arg164 in F1 structure.
3.2.3 FscJ Ferric-Siderophores Interaction
ITC supports FscJ binding with ferric-fuscachelin A with high affinity. FscJ was
titrated against ferric-fuscachelin A and an overall binding was modeled as an enthalpy
driven process with ΔH= -4.0 kcal mol-1 and a calculated dissociation constant of 5.4 μM
(Figure 3-4A). Mutation of the critical negative charged amino acids (E279A/N280A or
E302A/D310A) near the central cleft greatly decreased the FscJ ferric-fuscachelin A
73
binding ability (Figure 3-4B, C). Other ferric-siderophores include ferric-EDTA, ferric-
citrate, ferric-2,3-dihydroxybenzoic acid (DHB) and ferric-enterobactin were also tested
as putative substrates of FscJ. However no significant binding affinity was observed.
Figure 3-4. ITC binding analysis of FscJ (hexa-Histag free) against purified ferric-fuscachelin A complex. A) Wild type FscJ; B) E279A/N280A mutant and, C) E302A/D310A mutant.
Table 3-2. Protein structure comparison using the Dali server. FscJ monomer (P41-A) was used as the search model and top results are shown according to the Z-score and alignment root-mean-square deviation (RMSD, Å)
pdb Z-score RMSD lali Res. %ID Siderophore (Type)
3tny-A 23.0 3.4 251 280 28 Schizokinen (Hydroxamate)
4fkm-A 21.3 3.4 247 256 19 Ferrioxamine-B (Hydroxamate)
4b8y-A 20.9 3.7 250 277 22 (Hydroxamate)
3lhs-A 20.7 3.9 258 291 21 Staphyloferrin A (Hydroxamate)
3mwf-A 20.7 3.8 250 292 26 Staphyloferrin B (Hydroxamate)
3be5-A 20.7 3.8 255 294 24 (Unknown)
3eiw-A 20.6 4.0 260 292 20 Staphyloferrin A (Hydroxamate)
74
3.3 Structural Features and Siderophore Interaction Mechanism
3.3.1 FscJ Structural Comparison with Other Type III PBPs
FscJ shares a low structure homology to other substrate binding proteins in this
large protein family. A structure homology search (Dali server) shows that the top ten
most similar proteins have between 17%-28% sequence identity and a RMSD above
3.4 Å to FscJ (Table 3-2)131. Among the top structures, a number bind hydroxamate-
type siderophores ligands, including YfiY from B. cereus (PDB entry 3TNY, sequence
identity 28%)169 and SirA from S. aureus (PDB entry 3MWF, 26%)170. Heme-binding
proteins also share the topological similarities to FscJ with a common N-domain and a
similar large cleft located at the domain interface, among these are IsdE from S. aureus
(PDB entry 2Q8Q)171 and S. dysenteriae’s ShuT (PDB entry 2R7A)172. In contrast, FscJ
does not appear similar to free ion binding proteins belonging to type III PBP
family173,174. In order to confirm a lack of free metal binding, we employed single ion
soaking and no observable anomalous signal was present in crystals soaked with ferric
chloride, while diffraction data from an Hg derivative showed only an outer surface
interaction (HgI - His168). In neither experiment was there a resolved metal located in or
near the middle cleft binding pocket. Overall, FscJ shows the highest similarity to
uncharacterized proteins from N. alba and O. turbata with sequence identity 65% and
41% respectively, while other sequences in the current databases share an identity of
35% or lower (Figure 3-5). Interestingly, as compared to other homologs, FscJ has an
extended N terminus with an extra ~40 amino acids predicted to be intracellular of the
transmembrane helices. Similar to PBP TM0322 in Thermotoga maritima175, soluble
FscJ forms a strong dimer in solution. The stable dimers are hexa-Histag independent,
and the physiological roll of FscJ assembly is unknown. We postulate that this may
75
related to the in situ substrate-binding/ABC transporter system, which could possess
two substrate-binding sites per functional complex176.
Figure 3-5. FscJ sequence alignments with clustalW2. Top two non-redundant sequences (below 80% sequence identity) and top two sequences with structural information are shown aligned. FscJ residues are numbered as in full-length. Residues highlighted in yellow contains the Arg facing the ligand binding pocket, while residues highlighted in red are the negative charged amino acids locate on the edge of N- /C-domain cleft. Residues highlighted in blue are the hinged regions. Abbreviations: Tfu, Thermobifida fusca; Nal, Nocardiopsis alba; Otu, Oerskovia turbata; Bce, Bacillus cereus; Sau, Staphylococcus aureus.
3.3.2 pH-Dependent Dynamics Observed in Crystal Structures
We observed multiple forms of ligand-free FscJ in a similar crystallization
condition but with different pH values. FscJ conformations may represent the ‘open’ and
‘close’ state of the PBP with a hinged motion of 5.5° between F1 and F2 forms. (Figure
76
3-2B) The binding pocket of FscJ tends to ‘open’ for ligands binding with the pH
incensement. This observation could be relevant to iron acquition as the free ferric
concentration usually negatively corresponds to the environmental pH. FscJ is located
on the outer membrane of Gram-positive bacteria and has a direct environmental
contact. As a consequence, it may be favourable for FscJ to be in an open ligand
binding conformation over an extended pH range. The domain rotation region is located
between amino acids 224-228, on the end of long hinge α6. The hinged α6 consists
primarily of highly charged amino acid residues with negative Glu residues pointing
outward to bulk solvent (Figure 3-5, boxed in green). This charge arrangement may help
to immobilize the hinge region; especially the portion predicted to be closest to the cell
membrane177.
3.3.3 FscJ Has a Large Binding Pocket with Unique Charge Arrangement
Extensive efforts were made to cocrystallize various ligands with FscJ including
ferric-fuscachelin-A with and without a hexa-Histag. Unfortunately, we can only obtain
diffracting crystals with tagged FscJ and the structure shows the poly-histidine is bound
in the ligand pocket. Additionally, soaking of FscJ crystals with ligands failed to yield any
diffraction quality crystals. As all the prospective ligands are heavily charged and have
relatively a large size, the protein-ligand interaction likely disrupts the crystallographic
packing resulting in isomorphous crystals. ITC confirmed that FscJ binds with ferric-
fuscachelin A with an affinity of 5.4 μM. Fuscachelin A is the product from NRPS
biosynthesis pathway located in the same gene cluster with fscJ and likely is the
primary substrate for FscJ. FscJ shares a highest sequence identity with proteins that
transport six-coordinated hydroxamate type siderophores (YfiY and SirA). A structural
77
comparison of these and other homologues indicate that siderophores are bound in a
similar binding pocket (Figure 3-3). One prominent, common feature is a highly
conserved arginine (Arg164 in FscJ) located on the flexible loop between β2 and α4.
The residue is close to the ligated ferric ion and could help to balance the negative
charges of hydroxamate type ferric-siderophores. Compare to YfiY or SirA, FscJ has a
wilder binding pocket with N-terminal α2β2α4 and C-terminal β4β5β6 moving towards
outside (Figure 3-3). The larger pocket can accommodate ferric-fuscachelin A, a
relatively large siderophore. Meanwhile, Glu279, Glu302 and Asp310 on flexible loops
are conserved in uncharacterized proteins from N. alba and O. turbata, which could bind
similar siderophore compare to fuscachelins, but appear neither in YfiY nor SirA. The
negative charged glutamic/aspartic acids locate on the edge of siderophore binding
pocket will participate in the stabilization of positive charged D-arginines group on ferric-
fuscachelin A complex. Meanwhile, there is no negative charged amino acid residues
identified near YfiY/SirA substrate binding pocket. Neither YfiY nor SirA interact with
siderophores with large arginine subunits.
Docking attempts suggest that the siderophore binding occurs in the middle cleft,
as expected (Figure 3-6). Positive charges on siderophore D-Arg subunits are stabilized
through electrostatics/hydrogen bond interactions between Glu302/D-Arg1 (FuscA),
Asp310/D-Arg1 and Glu279/D-Arg2 (FuscA). The proposed binding site was surrounded
by hydrophobic residues including Phe281, Trp258, Phe244, Val246 and Ala282.
Arg164 on the N-domain faces the center of the pocket helping to stabilize the negative
charge on siderophore N-hydroxycarboxamino group. Overall, the ferric-
fuscachelin/FscJ interaction has a surface area of 1080 Å2 and predicted binding
78
constant of 6.6 μM consistent with our measured binding affinity (5.4 μM). Two sets of
mutagenesis were designed to confirm the critical electrostatics protein-ligand
interaction observed in the docking experiments. Mutant 1 contains E279A/N280A to
probe residues proposed to interact with D-Arg2 (Figure 3-6); mutants 2 (E302A/D310A)
examine side chains that stabilize D-Arg1. All protein mutants have a significantly
decreased or diminished binding affinity against ferric-fuscachelin A, which further
confirmed the essentiality of these negatively charged amino acids located on the edge
of FscJ binding pocket (Figure 3-4).
Figure 3-6. Molecular docking of FscJ with ferric fuscachelin A. The positively charged siderophore binds in the middle cleft and forms specific interactions with Glu302/D-Arg1 (FuscA, orange), Asp310/D-Arg1 and Glu279/D-Arg2 (FuscA, blue) interaction.
79
3.3.4 Conclusions and Insights
The chemistry and biology of metal binding siderophores is critical for microbe’s
survival and virulence. The ABC transporter system with its substrate specificity
commonly serves as the first ‘gate’ in the iron acquisition process. The siderophore
fuscachelin A of the moderate thermophile T. fusca is a unique siderophore with
uncommon molecular architecture. We are exploiting the production and utilization of
fuscachelin is a model system to study the specific iron acquisition process in Gram-
positive bacteria. The presented structure and biochemical characterization of type III
SBP FscJ provides insight into the complex molecular mechanisms of siderophore
recognition. Common to other family members, FscJ contains a large cleft for
fuscachelin recognition. Observed multiple conformations of FscJ provide insight into a
dynamic substrate binding mechanism. Unique binding models are utilized by FscJ,
including the recognition of a positively charged siderophore and the highly negative
charged hinge region facilitating subdomain movement. Additionally, FscJ contains an
unusually long mobile N-terminus of FscJ, that could be exploited in efforts to engineer
SBPs as biosensors178. Overall, the structure of FscJ helps understand the siderophore
recognition mechanism in gram positive bacteria, and allow unique insights that could
further facilitate the development of drugs that deliver through the iron acquisition
system in pathogen treatments88.
3.4 Materials and Methods
3.4.1 Cloning, Expression and Purification of FscJ
The gene for fscJ was amplified by using PCR from T. fusca genomic DNA with
the primers fscJ_N87 (5’-GCG GGATCC ACCGTCGAGATCCCCGCT), and fscJ_Cstop
(5’-GCG AAGCTT TCATAGGCCCTTGAGGAC) with N-terminal secretion signal and
80
lipidation site excluded (residues 1-86). The PCR product was treated with
corresponding endonucleases and then ligated into the expression vector pET28a.
Sequencing of several independent clones indicated that our construct of fscJ from T.
fusca ATCC 27730 deviates from the published genome of T. fusca strain YX at one
codon but did not change the encoded amino acid: TCT (Ser82) in ATCC 27730 while
TCC in T. fusca strain YX. The plasmid was transformed into E. coli BL21(DE3)pLysS
cells for overexpression. Cultures (Luria Broth, 1 L) were grown to OD600 = 0.8 at 37°C.
Overexpression was initiated by the addition of 400 μM isopropyl β-D-1-
thiogalactopyranoside (IPTG) and continued for 16 h at 25°C before harvesting by
centrifugation. Cell pellets were resuspended in 25 mL 0.5 M NaCl, 20 mM Tris-HCl, pH
7.5, and lysed at 14,000 psi through a microfluidizer (M-110L Pneumatic). The lysate
was then centrifuged at 15,000g for 20 min at 4°C. FscJ protein was purified through
immobilized metal affinity chromatography (IMAC). The supernatant was incubated for 1
h with 1 mL Ni-NTA Histag affinity resin (HisPur Ni-NTA resin, Thermo Scientific). The
bound resin was washed with 4 × 10 mL of 0.5 M NaCl, 25 mM imidazole, 20 mM Tris-
HCl, pH 7.5, and protein was eluted with 3 × 2 mL of 0.5 M NaCl, 250 mM imidazole, 20
mM Tris-HCl, pH 7.5. The elution fraction was further purified with gel filtration
chromatography (HiLoad 16/60 SuperDex-200 column, AKTA FPLC System, GE
Healthcare, 100 mM NaCl, 20 mM Tris-HCl, pH 7.5) before crystallization.
Selenomethionine (SeMet)-labelled protein was produced using metabolic inhibition of
methionine biosynthesis as described179. Cells were cultured in 1 L M9 minimal media
and grown to OD600 = 0.6 at 37°C. 60 mg L-SeMet was added to the culture 20 min
before cell induction and overexpression was initiated by adding 200 μM IPTG followed
81
by growth for 16 h at 25°C. The purification for Se-Met labelled protein was the same as
described above for the native protein with the addition of 10 mM dithiothreitol (DTT) to
the gel filtration purification buffer.
3.4.2 Cloning of Full Length FscJ, Expression and Localization
Full-length fscJ (fscJ_F) was amplified by using PCR from T. fusca genomic DNA
with the primers fscJ_N1 (GCG GGATCC ATG GGG TTG GGA AAG) and same
fscJ_Cstop. FscJ_F was expressed under similar conditions as the truncated proteins. 1
L Cell lysate was centrifuged at 10,000g for 2 h and the pelleted inclusion body
discarded. Cellular membrane was harvested with an additional 1 h centrifugation at
25,000g. The membrane was washed with 800 mM NaCl, 20 mM HEPES, pH 8.0, and
resuspended in 120 mM NaCl 20 mM HEPES, pH 8.0, with 0.05% dodecylmaltoside.
FscJ_F was purified through a Ni-NTA based IMAC method. Histag Western Blot was
used to confirm the expression; purified proteins from the membrane fraction were
analyzed with SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF)
membrane (Bio-Red). Poly-histidine monoclonal antibody (Sigma) was used as the
primary antibody and goat anti-rabbit IgG-HRP was used as the secondary antibody.
Western Blot signal was developed with SuperSignal West Pico Chemiluminescent
Substrate (Thermo Forma) and detected on a KODAK RP X-OMAT film.
3.4.3 Isothermal Titration Calorimetry Binding Affinity Determination
FscJ-siderophore binding affinity was determined with isothermal titration
calorimetry (ITC, MicroCal iTC200 system, Malvern). For binding studies, the hexa-
Histag was removed: 1 μL of thrombin (1 U L-1, Novagen) was mixed with 10 mg of
protein in 1 mL thrombin cleavage buffer (150 mM NaCl, 2.5 mM CaCl2, 20 mM Tris-
HCl, pH 8.4) and incubated for 16 h at room temperature. Cleaved protein was further
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purified using size exclusive chromatography (as described above). Ferric-fuscachelin A
was prepared from the isolated natural product and purified to homogeneity as reported
before77. 200 μL of 25 μM FscJ (0.025 mM) in 100 mM NaCl, 20 mM Tris-HCl, pH 7.5
was placed in the ITC reaction cell and 40 μL ferric-fuscachelin A (0.5 mM) was titrated
into the protein solution over time at 25°C. Heat change during the reaction was
detected and recorded for binding affinity calculation. Other protein ligands interaction
determinations were performed in a similar fashion. All titrations were repeated in
triplicate. The binding parameters and corresponding error are calculated using the
Origin software package155.
3.4.4 FscJ Crystallization and Optimization
FscJ initial crystal screenings were performed with vapor diffusion sitting drop
format (CrystalQuick 96 well sitting drop plate, Hampton Research). Small ‘angel hair’
like crystals were identified in the condition containing 0.2 M ammonium acetate, 0.1 M
sodium acetate, pH 4.6 and 30% w/v polyethylene glycol-4,000. Optimization of salt
concentration and pH were carried in vapor diffusion hanging drop format with
microseeding (24 well VDX crystallization plate, Hampton Research). 2 μL of protein (10
mg mL-1) solution plus 2 μL of precipitant were mixed and balanced against 1 mL of
reservoir solution at 25°C. Resultant rod-shaped single crystals were obtained in 0.16 M
ammonium acetate, 28% w/v polyethylene glycol-4,000 in and 0.1 M pH buffer at pH 4.6
(sodium acetate), 5.6 (sodium acetate), 6.5 (MES) and 7.5 (HEPES). Se-Met labelled
protein was crystallized in pH 4.6 conditions with same salt and precipitant as above. To
detect the possible metal/FscJ interaction, FscJ protein crystals were soaked in ferric
chloride or mercury acetate (1 mM) at pH 4.6 for 24 h. Crystals suitable in size and
83
shape were harvested and flash frozen in liquid nitrogen using 15% v/v glycerol as
cryoprotectant.
3.4.5 FscJ Atomic Structure Determination and Refinement
FscJ native and SeMet single-wavelength anomalous diffraction (SeMet-SAD)
data sets were collected at LS-CAT 21-ID-G beamline, Advanced Photon Source (APS),
Argonne National Laboratory (ANL) at a wavelength of 0.9786 Å (MARMOSAIC300
detector, 100 K). A mercury soaked data set was collected at GM/CA 23-ID-D beamline,
APS, ANL at a wavelength of 1.0064 Å (Pilatus3 6M, 100 K). Native data sets were
processed with XDS package156 into the space group I222, P21 or P41, with two, four or
two molecules per asymmetric unit, respectively. The SeMet-SAD data set was
processed into the space group I222. Four Se sites per asymmetric unit were found
using PHENIX.HYSS180,159 and phases were calculated with PHASER157. Initial rounds
of model building and refinement were completed using BUCCANEER181,182, COOT160
and REFMAC5183. The resultant poly-alanine model along with the diffraction data were
processed with six rounds of SAD-MR184. The location of heavy metals were further
refined with MLPHARE185 and PIRATE186, density maps were improved with
RESOLVE187, while model building was carried with SHELXE188 and BUCCANEER
sequentially. Native data sets were solved using molecular replacement with the model
from SAD-MR. Structures in space group I222, P21 and P41 were modeled and refined
independently with ARP/wARP189 and PHENIX.REFINE158. Hg ions in Hg-soaking data
set were located using anomalous difference maps calculated with PHENIX.MAP.
Sigma-A weighted, simulated annealing composite omit maps were used to judge and
verify structures throughout refinement. Water molecules were inspected carefully in
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calculated electron density maps. Structural illustrations were prepared with PyMOL161
and LIGPLOT190 Crystallographic data and refinement statistics are summarized.
Table 3-3. FscJ MR-SAD phasing and building cycles
Cycle Built resn. # (BACANNEER) Rwork/free
0 (poly-Ala) - 0.4399 / 0.4951
1 474 0.3859 / 0.4873
2 522 0.3682 / 0.4834
3 537 0.3317 / 0.4593
4 546 0.3491 / 0.4653
5 549 0.3338 / 0.4303
6 556 0.3079 / 0.3859
3.4.6 Detailed FscJ SAD-MR Based Experimental Phasing
FscJ shares a low sequence identity compared with all other published homolog
structures (Table 3-2) and direct molecular replacement do not yield any potential
solution. FscJ has only two methionines in the total sequence of ~300 amino acids
which both located on the N-terminus of the protein, making standard SAD a challenge.
Harvested SeMet-FscJ crystals were screened for anomalous signal and the best data
set was indexed to 3.2 Å resolution in space group I222. The anomalous measurability
of the data set was 0.1 at 8.3 Å resolution and 0.02 at 6.2 Å resolution from
phenix.xtriage analysis. Se sites identification was initiated with PHENIX.HYSS at 6.6 Å
resolution and phases were calculated with an initial figure of merit of 0.38, which can
be improved to 0.64 with density modification at 5.6 Å. A poly-alanine model was built
manually into the 5.6 Å electron density map and combined model and heavy atom sites
yield an interpretable 4.5 Å map from PHENIX.AUTOSOL. Final 3.2 Å map was
achieved through combining the HA sites from 5.6 Å and model from 4.5 Å, and
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corresponding full-length poly-Ala model was built in Coot based on predicted protein
3D structure from Rosetta server (RMSD of 3.6 Å compare with the final structure). With
a relatively low overall resolution and inaccurate phase information, the poly-alanine
model can be refined to Rfree/work =0.44/0.49% and the assignment of the corresponding
sidechain is rather difficult. A detailed SAD-MR strategy adapted from EMBL-Hamburg
Auto-Rickshaw was used to improve the model. The Rfree value dropped below 40%
after six rounds of MR-SAD (Table 3-3), and the resultant model was applied into
molecular replacement for native data sets in higher resolution.
3.4.7 Modeling the FscJ/Ferric-Siderophore Interaction
Ferric-fuscachelin A was docked into the FscJ structure (P41-A) with AutoDock 4
to understand the binding mode191. Ligand ferric-fuscachelin A was fully optimized with
density functional B3LYP/6-31G in Spartan '08 (solvent: water, pH 7.0)192. The core
region of ligand was restrained as rigid body while the D-arginine arms remain flexible
during the docking. Standard docking genetic algorithms were used with a rigid protein
in a grid covering the entire central cleft region.
Table 3-4. Primers for FscJ site-directed mutagenesis
Mutant # Site(s) Primers
1 E279AN280A CAGCAGCCGGCGGGGgcggccTTCGCTGCCTTCTAC
2 E302A GTCATCTTCTACgcgACCGACGCCCAG
2 D310A CAGGAGAACCCCgccCCGTTCACCGAG
3.4.8 FscJ Siderophore Binding Site Mutagenesis
Mutations of non-conserved binding site residues in the FscJ C-domain were
carried out using the Q5 site-directed mutagenesis kit (NEB) following the
manufacturer’s instructions. Mutant 1 contained mutations E279A and N280A; mutant 2
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contained E302A and D310A. All complete mutagenesis structures are confirmed with
DNA sequencing. Primers designed for the mutagenesis are listed (Table 3-4). Protein
mutants were purified and prepared as above.
87
CHAPTER 4
PRECURSOR PROTEIN-DIRECTED PEPTIDE MACROCYCLIZATION IN A RIBOSOMAL PEPTIDE NATURAL PRODUCT BIOSYNTHETIC PATHWAY
4.1 RiPPs Biosynthesis and Microviridin Biosynthetic Pathway
The diverse biosynthetic pathways to natural product, secondary metabolites
produce enormous structural diversity and biological activity193,194. Natural products can
be divided into distinct classes based on their biosynthesis pathways and the origins of
building blocks, including: ribosomally produced and post-translationally modified
peptides (RiPPs), non-ribosomal peptides (NRPs), polyketides (PKs), saccharides,
terpenes, and alkaloids195. Members of the large and diverse family of RiPPs exhibit
therapeutically useful properties including antitumor, antifungal, antibacterial and
antiviral196. RiPPs are derived from short DNA-encoded precursor proteins, necessarily
composed of proteinogenic amino acids196. The precursor proteins contains an N-
terminal leader peptide region197,198 (or less common a C-follower peptide199), which is
disposed during maturation, releasing one or more core peptides. Enzymatic post-
translational modification of the core peptides install functional/structural motifs and
include, for example, dehydration, epimerization, heterocyclization, prenylation,
lanthionine formation and macrocyclization (≥ 6 amino acids)197,198. Post-translational
modification commonly requires the presence of a leader peptide, as the core peptide
region alone will not be recognized for post-translational modification200,201.
Genetic engineering of core peptides is an attractive and proven approach to
create libraries of biomedically relevant macrocyclic peptides, recent examples include
manipulation of lasso peptides and microviridins in the heterologous host E. coli112,202.
Lasso peptides, as indicated by their names, are peptides assembled as a threaded
lasso and target cell surface receptors111; while microviridins are depsipeptidyl toxins
88
inhibiting serine proteases in the nM range203. Engineered, optimized heterologous
expression can produce diverse RiPPs with a high yield, for example reconstruction of
the tru pathway in E. coli results in the production of diverse natural and designed
cyanobactins204.
Figure 4-1. Representative precursor peptides and gene clusters in microviridin biosynthetic pathways. A) Aligned precursor peptides have a strictly conserved region (PFFARFL) highlighted in green. Residues involved in the cyclizations are labeled in orange (lactonization) and blue (lactamization). B) The microviridin J biosynthesis gene cluster in Microcystis aeruginosa MRC contains the genes of mdnA-mdnC, with the notable absence of mdnD or mdnE-like genes, present in M. aeruginosa NIES298. Planktothrix agardhii microviridin biosynthetic gene cluster contains two genes (mvdE and mvdF) encode for two independent precursor peptides. Gene all7013 in Nostoc sp. PCC7120 encodes a putative microviridin precursor peptide with three sequential core peptide region following a leader peptide.
Microviridin L biosynthesis in M. aeruginosa NIES298 involves five clustered
genes (mdnA-E) (Figure 4-1)205. The gene mdnA encodes a 49-amino acid precursor
peptide with a 14-C-terminal amino acids representing the core peptide region of the
final natural product. MdnB and mdnC translate to two homologs of the ATP-grasp
ligases, consisting of 325 and 324 amino acids, respectively. This cluster also contains
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mdnD, encoding an N-acetyl transferase, and mdnE, encoding a putative duel-function
polypeptide protease and transporter205,206. The leader peptide of MdnA is prerequisite
in the microviridin biosynthetic pathways, with a strictly conserved PFFARFL motif key
to recognition by the two the ATP grasp macrocylases (Figure 4-1)207. MdnC and MdnB
condense MdnA to form a tricyclic MdnAΔ3 (MdnA -3H2O) intermediate, then the leader
peptide region of MdnAΔ3 will be cleaved by MdnE and capped by N-terminal
acetylation (MdnD)205. The order of bond formation in the three macrocyclization
reactions is well resolved, typified by in the investigation of microviridin K biosynthesis in
Planktothrix agardhii CYA126/8 (Figure 4-1)208. The first ATP-grasp ligase (MvdD,
homologous the MdnC) sequentially catalyzes the Thr/Asp and Ser/Glu lactonization,
and the intermediate is then modified by MvdC (homologous to MdnB) through the
Lys/Glu lactamization. Besides the six key amino acid residues directly involved in the
cyclization, the core peptide region (MvdE) does possess limited modification
tolerancy209. Next, the leader peptide of MdnAΔ3 is cleaved presumably by MdnE, and
the nascent modified core peptide is then capped with N-terminal acetyl group by
MdnD205. In the microviridin biosynthesis, the leader peptide is prerequisite, and its
strictly conserved leader peptide motif is key to post-translational modifications (Figure
4-1)207.
On the basis of domain similarity and secondary structure prediction, the two
macrocyclases in microviridin pathways are three-domain ATP-grasp proteins
exemplified by RimK, a ligase involved in poly-α-glutamic acid synthesis and ribosomal
S6 modification210. The ATP-grasp superfamily is characterized by a unique ATP-
binding fold that ‘grasps’ a molecule of ATP in between the C-domain and the central
90
domain211. Core peptide binding and cyclization is most likely catalyzed between the N-
domain and the central domain212. Several, long loop regions between the three
subdomains of ATP-grasp ligases increase the overall protein flexibility and establish
the basis for the recognition of structurally diverse substrates. Most ATP-grasp ligases
are dimeric in structure with some examples of tetrameric, dimers of dimers. For
example, LysX involved in the conversion of α-aminoadipate (AAA) to lysine, is a
recently structurally characterized member of the ATP-grasp superfamily, with
relevance to the microviridin cyclases213. LysX ligates AAA to the protein LysW, to
activate the δ-carboxyl group for conversion to the ε-amine. The tetrameric LysX
consists of two dimers interacting through the central domains and binds two LysW
proteins, each sandwiched between two LysX N-domains. Additional members of the
ATP-grasp family that have been structurally characterized include D-alanine/D-alanine
ligase214, biotin carboxylase215 and diphosphoinositol pentakisphosphate kinase-2216.
While MdnB and MdnC produce secondary metabolite microviridins, most ATP-grasp
ligases are engaged in the primary metabolite pathways.
The microviridin J biosynthetic gene cluster (Figure 4-2) identified in M.
aeruginosa MRC, contains the genes mdnA-mdnC, with the notable absence of mdnD
or mdnE homologs (Figure 4-1)202. Mature microviridin J is a toxin that causes lethal
molting disruption in Daphnia through inhibition of serine-type proteases217. Compared
with other microviridins, microviridin J contains a unique Arg at position 40 that
facilitates its binding to trypsin202,218. The two ATP-grasp ligases, MdnB and MdnC, in
M. aeruginosa MRC share a significant degree of sequence homology (40%) between
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each other, but little (< 20% sequence identity, see Figure 4-3) to known crystal
structures, limiting the generation of reliable homology models for either enzyme.
Figure 4-2. Biosynthesis of microviridin J. A) The microviridin J biosynthetic pathway in Microcystis aeruginosa MRC contains three gene products: MdnC and MdnB catalyze the macrocyclization of MdnA to generate the intermediates MdnAΔ2 and MdnAΔ3, respectively. The cleavage of leader peptide followed by an acetylation produces the mature microviridin J. The residues coupled by MdnC and colored red and likewise MdnB, blue. B) In vitro cyclization of MdnA with purified MdnC and MdnB enzymes. HPLC analysis shows separation of the precursor peptide and two macrocyclization intermediates (wavelength=220 nm).
92
Figure 4-3. Alignment of MdnC, MdnB and other reported ATP-grasp ligases. MdnB and MdnC belong to ATP-grasp ligase superfamily consisting of a tri-domain structure. Of deposited structures, LysX is most similar sharing sequence identity of 19.5% and 17.2% with MdnC and MdnB respectively, while MdnB and MdnC share a sequence identity of 40.1%. Key residues corresponding to ATP binding interactions are highlighted in red. The amino acid region highlighted in green corresponds to leader peptide recognition.
Cyclization of peptides is a common approach in both natural and synthetic
systems to generate biologically-active peptides. The cyclic structures are less
susceptible to in vivo degradation and proteolysis and present rigid conformations
entropically-favored to bind targets. . Several enzymatic strategies are known to
catalyze the cyclization of peptides including serine protease-type mechanisms in
nonribosomal219 and ribosomal peptide220,221 pathways along with ATP-dependent
activation paths202,222. These approaches often exhibit high degrees of specificity and
efficiency that can rival synthetic methods. Here we describe the structure and
biochemical characterization of the two peptide macrocyclases, MdnC and MdnB, from
the microviridin J gene cluster. The results provide a structural basis for the interactions
93
of peptide macrocyclases with their precursor peptide substrates, a common and
integral feature of pathways to ribosomal peptide natural products.
Figure 4-4. Expression and purification of MdnB and MdnC as dimeric proteins. A) MdnC and MdnB shares a similar retention time in size exclusion chromatograph and are predicted to be dimers in solution. Molecular weight standards (BioRad) were plotted as retention volume vs log (MW). B) MdnC and MdnB, with a molecular weight of ~35 kDa, run as dimers in native PAGE analysis (~80 kDa).
4.2 Leader Protein-Directed Microviridin J Biosynthesis
4.2.1 MdnB and MdnC Catalyze MdnA Cyclization
Full-length MdnB and MdnC were heterologously expressed in E. coli
independently as C-terminal hexa-histidine fusion proteins utilizing a short alanine linker
between the tag and the enzyme. As is common with the ATP grasp superfamily, size-
exclusion chromatography profiles and native-PAGE analysis indicate that both MdnB
and MdnC form homodimers (Figure 4). In vitro assays using purified MdnC and MdnB
showed complete cyclization of MdnA to the microviridin precursor in the presence of
Mg2+ and ATP. MdnC catalyzes two lactonizations to form MdnAΔ2, while MdnB installs
the final macrolactam to produce MdnAΔ3 (Figure 4-2, Figure 4-5 and Table 4-1).
Additionally, kinetic analysis of the MdnC catalyzed double-cyclization result in a
measured KM (MdnA) of 23.8±1.2 μM and apparent kcat of 0.47±0.02 min-1 at 25°C
(Figure 4-6).
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Figure 4-5. Analysis of full-length MdnA cyclizations. MdnA and its cyclic products have been identified in MS analysis. Refer to Table 4-1 for masses.
Figure 4-6. Kinetic analysis of the MdnC catalyzed dicyclization. A) Representative HPLC traces for MdnC-catalyzed MdnA dicyclization and initial rate determination. B) Michaelis-Menten plot created with various initial rates at different substrate concentrations. Each initial rate has been determined as triplets.
4.2.2 MdnA Leader Peptide Interacts with MdnB and MdnC Macrocyclases
A hallmark of ribosomal peptide biosynthetic pathways is protein/protein
interactions of precursor peptides with the catalytic enzymes through specific structural
elements. MdnA is 49 amino acids in length with 13 of those amino acids included in the
final natural product structure. To understand the interaction of MdnA with the
macrocyclases, we conducted ITC-based binding assays. Full-length MdnA (amino
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acids 1-49) binds MdnC, with a measured binding constant (KD) of 112±52 nM (Figure
4-7 and Table 4-2). This binding interaction is ATP-independent and the addition of the
stable ATP analog AMPPNP, or ADP does not change the MdnA/MdnC interaction
thermal profile. We next removed the core peptide region of MdnA to create a leader
peptide-only analog (MdnA1-35), and observed the similar level of interactions between
MdnA1-35 and MdnC (KD = 146±52 nM). These results indicate that the MdnA/MdnC
interaction is predominantly leader peptide-dependent, while the core peptide of MdnA
does not contribute significantly in the initial stage of substrate recognition. Using a
similar approach, we observed a weaker interaction between MdnB and full length
MdnA or MdnA1-35 (KD values were 4.8±1.0 and 2.8±0.8 μM, respectively). Because the
natural substrate for MdnB is the di-cyclo MdnAΔ2 and not MdnA, the reduced binding
affinities suggest that the modified core peptide likely contribute to the MdnB/MdnA
interactions.
Table 4-1. MS profiles of MdnA and MdnA variants
MS fragments (positive mode) Experimental Theoretical
+3 +4 +5 +6 +7 +8
MdnA 1430.3 1144.5 953.9 817.8 715.7 5717.5
5718.31
MdnAΔ1 1426.0 1140.8 950.8 815.3 5699.5
MdnAΔ2 1421.4 1137.3 948.0 812.8 711.3 5681.6
MdnAΔ3 1416.6 1134.1 944.8 810.2 5663.8
MdnA1-35 978.4 782.9 652.7 3909.8 3910.37
MdnAAc20-49 1189.4 892.3 714.1 3565.3 3563.76
MdnAAc20-49Δ1 1183.3 887.8 710.5 3547.2
MdnA11-49 1147.0 917.8 765.0 4584.0 4585.07
MdnA11-49Δ2 1138.1 910.7 759.1 4548.5
MdnA11-49Δ3 1133.6 907.0 756.0 4530.1
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Figure 4-7. ITC profiles of MdnA variants interact with macrocyclases MdnB and MdnC. Detailed fitting information is listed in Table 4-2.
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Figure 4-8. Overall structures of MdnC and MdnB. A) Two views of MdnB (blue/grey) and, B) MdnC dimers (orange/grey). MdnC forms a co-complex with the precursor peptide MdnA1-35 (green). C) Superimposition of the MdnC and MdnB protomers illustrate domain movement involved in precursor peptide binding. D) Evolutionary conservation map of MdnC protomer highlights conserved regions of the C-domain. The modeled ADP/Mg+2 binding site is noted on the map.
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Table 4-2. ITC-based precursor peptide/cyclase interaction
Protein Ligand Site(s) Kd
MdnC MdnA 0.95±0.02 112±52 nM
MdnC MdnA1-35 0.90±0.01 146±52 nM
MdnC MdnA11-49 1.04±0.01 308±78 nM
MdnC MdnAAc20-49 n.d.*
MdnC ATP n.d.
MdnCAA MdnA n.d.
MdnCKK MdnA n.d.
MdnCE293A/N295A MdnA 0.84±0.05 575±302 nM
MdnCD281A MdnA 0.71±0.01 568±80 nM
MdnB MdnA 0.80±0.10 4.85±1.02 μM
MdnB MdnA1-35 1.12±0.02 2.85±0.77 μM
MdnB MdnAAc20-49 n.d. * n.d. - not detected, with no observable heat change during the titration.
4.2.3 Overall Structure of MdnC and MdnB
To further understand the leader peptide prerequisition in the microviridin
biosynthetic pathway, we determined the X-ray crystal structures of MdnC and MdnB.
The phase information for both MdnC and MdnB was calculated from SeMet derivatized
proteins using SAD (Table 4-3). Multiple crystallization trials of MdnC and MdnB with
MdnA and/or ATP/nonhydrolyzable analogs all resulted in the same “active site free”
structures, with several unresolved isomorphous loop regions key to the enzymatic
reaction and no substrate or cofactor apparent in the electron density maps. To obtain a
more complete model, MdnA1-35 was chosen as the alternative substrate mimic to probe
the precursor peptide/enzymes interactions. MdnC was crystallized in the presence of
the C-truncated MdnA1-35 (without the core peptide region) and the structure of the
complex was solved. The MdnC/MdnA1-35 complex was in a tetragonal form, with four
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dimers per asymmetric unit. In contrast, only the apo-form of MdnB was obtained when
co-crystallized with MdnA1-35. The orthorhombic crystal form of MdnB consists of two
protomers per asymmetric unit, where the biological unit (dimer), is formed via
crystallographic symmetry. Compared with the circular octameric geometry of MdnC in
the crystal, MdnB dimers pack in a linear form (Figure 4-8 and Figure 4-9). Residues 2-
244/250-325 in MdnC (chain A) and 5-238/254-326 in MdnB (chain A) were successfully
built into the electron density maps. Residues 11-22 of MdnA (chain I) are resolved in
the MdnC/MdnA1-35 complex.
Figure 4-9. Crystallographic packing of MdnC and MdnB. MdnC crystal packs in space group P41, with four dimers per asymmetric unit. MdnB crystallize in space group C2, consisting of two protomers per asymmetric unit. Observed MdnA1-
35 bound with MdnC protomers in 1:1 ratio. Calculated mFo-DFc difference map corresponds for MdnA fragments are displayed in green at 2.0σ contour level.
Both MdnB and MdnC fit in the overall ATP-grasp ligase structural motif, with
three subdomains: N-domain, central domain and C-domain (Figure 4-8). MdnC and
MdnB form similar homodimer assemblies with an interface area of approximately 2748
100
Å2 (MdnC) and 2442 Å2 (MdnB) respectively. Comparison of the dimers of MdnC and
MdnB show an overall Cα RMSD of 1.7 Å. Amino acids near the dimeric interface are
predominantly hydrophobic in nature, while the ATP binding sites have a higher
calculated electrostatic potential (Figure 4-10). The dimeric interaction is mediated
through two key antiparallel β-strands between the N-domain (β3) and the neighboring
central domain (β’8) (Figure 4-11). These two strands are structurally conserved in all
ATP-grasp ligase family members. In addition, α3 extends along the neighboring central
domain, interacting with β’9 and contributing to the stabilization of the dimer. Compared
with other reported ATP-grasp ligases, the 23-amino acid long helix (α3) in MdnC/B is
atypical, as others commonly contain a much shorter helix at this position. Two helices,
α4 and α’4, connecting the N-domain and central domain, are located in the middle of
the dimeric interface. The α4 and α’4 in MdnC dimers are in a close contact, whereas in
the MdnB dimer they are ~6 Å apart, creating a top-to-bottom tunnel-like feature.
Figure 4-10. Electrostatics map of MdnC and MdnB dimers. Color-coded electrostatic surface map. MdnC and MdnB dimers are the same orientations as shown in Figure 4-8. A tunnel-like feature in MdnB dimeric interface has been observed. Hydrophobic residues are assembled near the dimeric interface, while hydrophilic residues are predominantly near the ATP binding site.
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Figure 4-11. MdnC crystallized as a dimer. The N-domain of MdnC (chain A) is in orange, while the neighboring central domain (chain B) is in grey. Key interactions between antiparallel β3/β’8 stabilize the dimerization. Other interactions include α3/β’9 and α4/α’4.
Figure 4-12. C-alpha distance difference plotting between MdnC and MdnB protomers. Plot (x, y) indicate the absolute difference between the distance of residue x Cα and residue y Cα in MdnC and MdnB. Amino acid movement between MdnC and MdnB protomers has been observed in β9β10 hairpin and helix α7 regions.
The MdnC and MdnB protomers are overall structurally similar, but there are
several differences in their central domains (Figure 4-8), specifically in the β9β10
hairpin region and the α7 region (Figure 4-12). MdnC possesses a long two-stranded
antiparallel β-sheet (β9β10), followed by a relatively ordered helix, α7. The β9β10 and
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α7 regions are anchored by the MdnA leader peptide. On the contrary, the β9β10
hairpin of MdnB lays on the C-domain, while the α7 region is disordered into a flexible
loop. In the two structures, the β9β10 hairpins are in significantly different
conformations, the turn of the hairpins are 25 Å apart comparing MdnC and MdnB.
Table 4-3. Data collection and refinement statistics of macrocyclases MdnB and MdnC
SeMet Derivative Native
MdnB MdnC MdnB MdnC+MdnA1-35
Data collection
Space group C121 P41 C121 P41
Cell dimensions
a, b, c (Å) 128.49, 36.13, 139.90
132.62, 132.62, 196.98
128.62 37.99 139.38
132.56, 132.56, 198.16
() 90, 116.9, 90 90, 90, 90 90, 116.6, 90 90, 90, 90
Resolution (Å) 45.2-2.51
(2.67-2.51)*
39.5-2.76
(2.81-2.76)
33.4-2.28
(2.36-2.28)
39.7-2.66
(2.71-2.66)
Rmerge 0.107 (0.66) 0.183 (0.80) 0.053 (0.41) 0.114 (0.67)
I / σI 18.7 (1.8) 19.4 (2.1) 17.0 (3.6) 17.1 (3.8)
Completeness (%) 88.3 (69.1) 99.8 (99.7) 99.9 (97.1) 99.9 (92.8)
Redundancy 4.6 (4.5) 12.5 (11.4) 4.9 (4.9) 11.9 (10.8)
Refinement
Resolution (Å) 33.4-2.28 39.7-2.66
No. reflections 137054 1155055
Rwork / Rfree 0.215 / 0.255 0.219 / 0.260
No. atoms 4433 20643
Protein 4376 19872
Precursor peptide - 771
Water 43 -
B-factors 47.82 55.97
Protein 47.81 55.16
Precursor peptide - 76.70
Water 48.60 -
R.m.s. deviations
Bond lengths (Å) 0.009 0.003
Bond angles () 1.02 0.64 *Single crystal was used for each data set;
*Values in parentheses are for highest-resolution shell;
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Figure 4-13. MdnC uses ATP to catalyze the macrocyclization of the precursor peptide
MdnA. A) Modelled ADP interacts with the C-domain and central domain of MdnC. The positions of Mg2+ and ADP are adapted from the aligned LysX structure (PDB entry 3VPD, 20% sequence identity). B) Interactions of MdnC with the precursor peptide MdnA. The strictly conserved region of MdnA (PFFARFL) is well-resolved in the electron density map. Helix α7 and β9β10 hairpin of MdnC sandwich the helical region of the resolved MdnA leader peptide. Calculated mFo-DFc map is displayed at a 1.5σ contour level.
Table 4-4. Key residues in nucleotide interaction
MdnC MdnB LysX RimK DDL Proposed interaction
Lys125 Lys131 Lys127 Lys100 Lys97 Binds to -phosphate of ATP
Lys166 Lys171 Lys87 Lys141 Lys144 Binds to -phosphate of ATP
Gln207 Gln211 Gln167 Glu178 Glu180 Hydrogen bond to adenosine N6
Glu215 Glu219 Asp176 Asp187 Glu187 Hydrogen bond to ribose O3’
Asp281 Asp284 Asp234 Asp248 Asp257 Metal ion coordination
Glu294 Glu297 Glu250 Glu260 Glu270 Metal ion coordination
Asn296 Asn299 Asn252 Asn262 Asn272 Metal ion coordination
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Figure 4-14. Characterization of determinants for MdnC catalyzed cyclization. A) Extraction of crude reaction mixture followed by mass spec analysis, relative ion intensity corresponding to ADP and AMP are plotted. MdnC produces ADP but not AMP during the catalysis. B) Mass spec analysis of product formation for MdnC and site-directed mutants. Masses (positive mode) are 1137:1138 for MdnAΔ2 and 1140:1141 for MdnAΔ1. Only the MdnCAA mutant produces cyclic product. C) MS for the MdnCAA reaction mixture, indicating the formation of MdnAΔ1.
Figure 4-15. MdnC central domain interacts with the precursor peptide MdnA. Alignment of the β9β10 hairpin and helix α7 regions among enzymes in microviridin biosynthetic pathways. D192 (dark red) is well conserved in MdnC-like and MdnB-like homologs. E191 is conserved in MdnC, MdnB and MdnB-like homologs, but is substituted as an aspartate in most other MdnC-like macrocyclases.
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4.2.4 Key Residues for Nucleotide Binding and Catalysis
The macrolactone cyclizations of Thr39/Asp45, Ser43/Glu47 (by MdnC) and
Lys41/Glu48 (by MdnB) require Mg2+ and ATP208. The reactions of MdnC and MdnB
both generate ADP, but not AMP (Figure 4-13 and Figure 4-14A). These results suggest
that the cyclizations are most likely to occur via acylphosphorylation, as opposed to
(also common) the generation of acyl-AMP intermediates. Unfortunately, as mentioned
and despite extensive efforts, we were unable to observed ATP, ADP or various non-
hydrolyzable analogs soaked or co-crystallized with MdnC or MdnB in the calculated
electron density maps. Also, unlike other ATP-grasp ligases, whose interactions with
ATP have been experimentally determined 213, neither MdnC nor MdnB show an
observable heat change with ATP (and its analogs) using ITC analysis. Nonetheless,
the ATP binding pocket of the ATP-grasp ligases is structurally conserved, allowing the
determination of the key residues in MdnC and MdnB that interact with the nucleotide
and facilitate the macrocyclization (Figure 4-13A and Table 4-4). Using structural
alignment, critical residues involved in ATP interactions and metal ion coordination
include Lys125, Lys166, Gln207, Glu215, Asp281, Glu294 and Asn296 (for MdnC). To
probe the importance of these residues in the MdnC reaction, we mutated each to
alanine. The mutation of any of these residues to Ala (K125A, K166A, Q207A, D281A
and E194A/N296A, Figure 4-14) completely abolishes the MdnC cyclization activity.
These residues are predicted to be primarily involved in ATP binding and not interaction
with the precursor peptide. Indeed, representatives of these MdnC mutants (D281A and
E294A/N296A) showed the similar level of interaction with the precursor peptide MdnA
as measured with ITC (Figure 4-7).
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4.2.5 Recognition of MdnA Leader Peptide by MdnC
Our binding measurements suggest that the MdnA interacts with MdnC in a core
peptide-independent manner (Figure 4-7 and Table 4-2). Consistent with this, amino
acids 11-22 of MdnA1-35 were well-resolved in the calculated electron density map of the
MdnC/MdnA1-35 complex. This region of the leader peptide includes the strictly
conserved motif of PFFARFL bound in an α-helix conformation (Figure 4-13B)207.
MdnA1-35 interacts with the central domain of MdnC through electrostatic interactions
between Arg17 of MdnA and Glu191/Asp192/Asn195 of MdnC α7. In most other MdnC-
like macrocyclases, Glu191 is replaced by an aspartate residue, while Asp192 is well
conserved among all microviridin macrocyclases (Figure 4-15). Meanwhile, electrostatic
interactions between the backbone amides of Ser20 (MdnA) and Val182 (MdnC)
induced the movement of MdnC β9β10 hairpin toward MdnA1-35. The interface area of
bound MdnA1-35 and MdnC is ~740 Å2.
To probe the key residues of MdnC in binding to MdnA, we created two MdnC
variants with double mutations, MdnCE191K/D192K and MdnCE191A/D192A (referred to as
MdnCKK and MdnCAA, respectively). The charge reversal of the conserved Glu191 and
Asp192 residues completely abolished the MdnA/MdnCKK interaction (Figure 4-7), and
MdnCKK had no macrocyclization activity toward the full length MdnA. On the other
hand, the more conservative mutant MdnCAA retained partial cyclization activity but had
no detectable MdnA/MdnCAA binding interaction via ITC analysis. Compared with WT
MdnC, MdnCAA exhibited a significantly slower reaction, and promoted the formation of
MdnAΔ1 and MdnAΔ2 in a ~1:1 ratio (Figure 4-14). These observations confirmed the
roles of Glu191 and Asp192 and α7 in precursor protein recognition.
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Figure 4-16. Binding and cyclization activity of macrocyclases toward MdnA variants. Truncated MdnA variants were utilized to probe the binding and activity determinants of MdnC and MdnB. For binding, ++ represents strong binding and +, weak binding (see Figure 4-7 and Table 4-2). Cyclization is indicated by a ‘+’ or ‘-’ for no observed reaction. n.d. = no activity detected.
Figure 4-17. MdnA variants and their macrocyclizations. A) MdnA11-39 can be recognized and processed by both MdnC and MdnB to form the tri-cyclic product while, B) MdnAAc20-49 alone is not an active substrate for MdnC. MdnC can be constitutively activated with the presence of MdnA1-35, and catalyze the single cyclization of MdnAAc20-49. However, the single cyclic product in in trans reaction cannot be further converged into di- (or tri-) cyclo product, neither with an extended reaction time (data not shown), nor with an increased MdnA1-35 concentration.
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To further evaluate the importance of observed MdnA structural elements in the
enzymatic macrocyclization chemistry, we created two truncated precursor protein
variants and characterized their functions (Figure 4-16). MdnAAc20-49 (amino acid 20-49
with an N-terminal acetyl group, Figure 4-16, line iv) does not include the conserved,
structurally ordered α-helix region, while MdnA11-49 (Figure 4-16, iii) contains the α-helix
but ten amino acids at the N-terminus of MdnA, which were not observed in the
MdnC/MdnA1-35 complex, are removed. As predicted, MdnAAc20-49 showed no binding to
MdnC and was not cyclized in our in vitro assays (Figure 4-17). On the contrast, the
MdnA11-49 variant bound to MdnC with comparable parameters as full length MdnA and
could be di-cyclized (Figure 4-7and Figure 4-17A). Additionally, the reaction product of
MdnC, MdnA11-49Δ2, was further converted into MdnA11-49Δ3 by MdnB. These results
along with the crystal structure have revealed the minimum requirements for efficient
precursor peptide binding and cyclization.
Next, we explored the potential of leader-free macrocyclization following similar
approaches used in other RiPPs biosynthetic pathways223,224. We attempted in trans
cyclization of an ‘inactive’ substrate (MdnAAc20-49) with the α-helical MdnA leader peptide
MdnA1-35. Indeed, the combination of ‘inactive’ MdnAAc20-49 with the inducer MdnA1-35 led
to the production of a cyclic intermediate. Unexpectedly, conversion to a single
cyclization product was observed (MdnAAc20-49Δ1) and the product of the second
cyclization was not detected. Identical results were obtained employing extended
reaction times or increased MdnA1-35 concentration. MdnAAc20-49Δ1 was not a substrate
of MdnB in the presence or absence of inducer MdnA1-35 (Figure 4-17B).
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4.3 Structures of MdnC and MdnB Facilitate to Understand the Precursor Recognition in RiPP Pathway
Post-translational modification of ribosomal precursor peptides allows for the
production of large libraries of natural (and synthetic) variants. Macrocyclization is one
of the most common modifications in RiPPs and other natural product classes, including
nonribosomal peptides and polyketides. The cyclic architecture contributes to the
structural and functional diversity, as well as the rigidity and in vivo stability of the final
product. The well characterized patellamide (RiPP) biosynthetic pathway utilizes a
subtilisin-like serine protease domain (PatG) to generate the mature cyclic peptide
products220. PatG recognizes the linear precursor peptide PatA, and directs peptide
cyclization through serine acyl-enzyme intermediate chemistry. A similar Asp-His-Ser
catalytic assembly and mechanism is utilized by nonribosomal peptide synthetase
(NPRS) thioesterase domains219. The microviridins and marinostatins are structurally
complex ribosomal peptides with multiple macrolactam/lactone cyclizations, formed by
condensation of amino acid side chains and/or termini198. This class of RIPPs is
produced by cyanobacteria and exhibits serine protease inhibition leading to cytotoxic
effects on neighboring organisms217. The microviridin J gene cluster of M. aeruginosa
MRC contains a minimal set of genes (mdnA-C) for the biosynthesis of microviridin,
making it an excellent model system for the study of RiPP biosynthesis and cyclization.
In this report, we characterized two macrocyclases in the microviridin J biosynthesis
pathway (MdnC and MdnB), along with the precursor protein MdnA. The in vitro
catalytic activity of MdnC and MdnB has been demonstrated, and the crystal structures
of both enzymes have been solved. Both enzymes belong to the ATP-grasp family
whose members utilize a wide range of chemistries with ATP-based activation in
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common, and represent a distinct strategy for peptide cyclization. The general
mechanism of the ligations has been dissected in similar systems and has been shown
to utilize ATP to activate the carboxylate substrates to yield an acylphosphate
intermediate and ADP212. A distinct structural feature of the two macrocyclases is the
presence of several long interdomain loops that show significant disorders in the
electron density maps. Similar to many ATP-grasp family members, this feature likely
provides the interdomain flexibility necessary for the binding of the 13-amino-acids core
peptide substrate.
MdnC catalyzes the first step in the post-translational modification of microviridin
J by the formation of two macrolactone rings (Figure 4-2, MdnA to MdnAΔ2). Compared
with the ‘apo-form’ of MdnB, MdnA bound MdnC exhibits a large movement of the
central domain. The ~25 Å shift of the β9β10 hairpin region in MdnC opens the
interdomain pocket, and allows the core peptide to access bound ATP between the
central domain and C-domain. Meanwhile, crystal structure of MdnB represents the
‘closed’ conformation with the β9β10 region blocking the pocket. Binding of the MdnA
leader peptide of MdnA restrains the β9β10 hairpin and the α7 helix in MdnC. These
regions may act as allosteric sites for subsequent core peptide recognition and
catalysis, assisting in orienting the inherently disordered active site. In support of this
hypothesis, mutagenesis of conserved residues on α7 significantly alters the enzymatic
efficiency.
The structural basis and mechanism of precursor peptide recognition is the key
factor in enzyme/substrate selectivity in RiPP pathways. Only limited structures of RiPP
biosynthetic enzymes have been determined in complex with their corresponding
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precursor peptides, and include dehydratase NisB involved in nisin biosynthesis225 and
heterocyclase, LynD, involved in cyanobactin biosynthesis224. Both enzymes use a
three-stranded antiparallel β-sheet to interact with the conserved regions of the leader
region of the precursor peptide. An RiPP precursor peptide recognition element (RRE)
has been predicted based on these partial co-complex structures and related
mutagenesis studies198,226,227. By comparison, MdnC and MdnB do not possess a
similar RRE. The interaction between MdnA and the two ATP-grasp ligases represent a
novel type of RiPP precursor peptide recognition mechanism. Most leader peptides tend
to contain α-helices in solution, and presumably maintain this structure when bound to
the biosynthetic proteins197. Structural comparison between MdnA-bound MdnC and
apo-MdnB indicates that the α-helical leader peptide is critical for the conformation
change of the biosynthetic enzymes, while the conformational change is the key factor
modulating the enzyme activity. While the α-helix is important for post-translational
modification machinery, the first 10 amino acids at the N-terminus of MdnA (1-10) are
not required for precursor peptide recognition. Similar substrate specificity has been
observed in the lacticin biosynthetic pathway, where a propeptide LctA variant with the
deletion of its N-terminal eight amino acids could be fully processed200. In addition,
compared with MdnB, the MdnC/leader peptide (MdnA1-35) binding interaction is ~10
fold tighter. The differential binding affinities of the precursor peptides can be
rationalized in several ways. It is reasonable to predict that the linear MdnA peptide is
less stable and thus is required for it to be processed rapidly to the macrocycle. Also the
substrate for MdnB is the di-cyclized core peptide and it is likely that both the leader
peptide and the cyclized core contribute in binding and recognition by the second
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enzyme, MdnB. Likewise, distinct leader peptide recognition may help to avoid the
competitive inhibition of MdnB by the precursor peptide.
In trans activation of the macrocyclase by the helical leader peptide alone led to
an active enzyme, an interesting feature of the a few RiPP systems studied.
Unexpectedly, MdnC in trans processing of the leader-free MdnAAc20-49 with MdnA1-35
only produced a product with a single cyclization. In comparison, LctM catalyzes
multiple dehydrations of leader-free LctA with a high concentration of leader peptide223.
The full length MdnA may also facilitate the movement (and rotation) following the first
cyclization of the core peptide, thus facilitating the second cyclization. This process
might require leader peptide and core peptide stay bound. A trapped MdnAΔ1 in the
MdnCAA mutant could inhibit the dynamics leading to the observation of no further
processing.
The helix of the MdnA leader peptide is ~15 amino acids away from the core
peptide of MdnA. This peptide region was unresolved in MdnC/MdnA1-35 complex
structure, and may be disordered and/or flexible. However, simple modelling suggests
the antiparallel β-strands (β9β10) in between the leader peptide and the ATP active site
could interact in a β-sheet motif in a similar fashion as of the PatE/LynD interaction224.
The MdnA α-helical C-terminus might extend along MdnC β10 near the MdnC dimeric
interface, and then fold back to the active site between MdnC N-domain/central domain,
where the core peptide could approach bound ATP for activation (Figure 4-18). Another
member of the ATP-grasp ligase, LysX uses the N-domain to interact with β-barrel
LysW. The C-terminus of LysW extended into LysX active site213. On the contrast, the
N-domain of both MdnC and MdnB show significant differences compare with LysX N-
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domain (Figure 4-3). One of the structural differences between MdnB and MdnC is the
tunnel-like feature of MdnB between at the dimeric interface. This larger central cavity
could be necessary for binding and processing of its more rigid substrate, MdnA∆2.
We were not able to observe a complex structure of MdnC or MdnB with the
MdnA core peptide (the region cyclized and in the final product), nor did we observe any
core peptide binding interaction with MdnC or MdnB by ITC. This suggests that the
macrocyclase-core peptide interaction is not the key determinant for binding/recognition
and the interaction between the macrocyclase and the leader peptide plays a more
significant role. Indeed, several reports have shown that alteration of the core peptide
region is tolerated whereas changes to the conserved region of the leader peptide are
not207. This is also consistent with the natural, genetic variability observed in microviridin
gene clusters. In the P. agardhii gene cluster, mvdE and mvdF represent two distinct
propeptides that are both substrates for the macrocyclases (Figure 4-1). Additionally,
there are several examples of a single precursor peptide gene product containing one
leader peptide and multiple core peptides. The all7013 in Nostoc sp. PCC 7120
contains the strictly conserved α-helical element in the leader peptide region and three
different core peptide regions, indicating the potential to form three different final
products (Figure 4-1). Similar combinatorial assembly in RiPP systems has been
detailed in the patellamide biosynthetic pathway228.
The structural basis of substrate precursor peptide binding in the microviridin
biosynthetic pathway is summarized in Figure 4-18. The α-helical region of the leader
peptide binds to the α7 of macrocyclases and induces a conformational change of the
β9β10 hairpin, which allows the core peptide region to access the ATP binding pocket
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for activation and cyclization. These processes require the presence of the helix
element on leader peptide, but the amino acids at the N-terminus to this helix region are
not required for the macrocyclizations. The disruption of leader peptide/macrocyclase
interaction by mutating the key residues on α7 also abolishes the cyclization reactions.
In addition, a constitutively activated macrocyclization can be observed through an in
trans reaction. The results presented provide novel insights into the understanding of
RiPP biosynthetic pathways and facilitate discovery and engineering of RiPPs
pathways.
Figure 4-18. Leader peptide directed peptide macrocyclization in the microviridin J
biosynthetic pathway. A) The leader peptide of MdnA activates macrocyclation by orienting α7 and by inducing a large shift of the β9β10 hairpin. B) The α-helical element of the precursor peptide, as well as residues on α7 (Glu191/Asp192, refers as to ‘ED’), are critical and removing these elements result in no cyclizations, C) whereas the N-terminus of the substrate is not requisite for either MdnB or MdnC macrocyclizations. D) In trans activation of MdnC using the α-helical element of the precursor peptide and an inactive substrate produces a partially active macrocyclase. E) Model of the interactions of full length MdnA (green) with the macrocyclase, MdnC. The cyclized core peptide region was adapted from pdb entry 4KTU and docked into MdnC with modelled ADP.
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4.4. Materials and Methods
4.4.1 Protein Cloning, Expression and Purification
Codon-optimized, full-length mdnB and mdnC (M. aeruginosa MRC) were
purchased from Mr. Gene (Regenburg, Germany) supplied in pMA vectors. mdnB and
mdnC were amplified using PCR (Table 4-5) and a short C-terminal AAAHHHHHH
hexa-histidine tag was encoded in the C-terminal primers bearing the Xhol restriction
site for both MdnB and MdnC. The mdnB and mdnC genes were then ligated into the
expression vector pET30a. MdnC was expressed in E. coli BL21(DE3) pLysS. Cultures
(1 L) were grown at 37°C to an OD600=0.6 and overexpression was initiated by adding
isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration 100 μM). Growth was
continued for 12 h at 18°C, before the cells were harvested by centrifugation. Cell
pellets were resuspended in 25 mL of 0.5 M NaCl and 20 mM Tris-HCl, pH 7.5, and
lysed at 14,000 psi through a nitrogen-pressure microfluidizer cell (M-110L Pneumatic).
The lysate was clarified by centrifugation at 15,000g for 20 min at 4°C. MdnC was
purified by immobilized metal affinity chromatography (HisPur Ni-NTA Resin, Thermo
Scientific). After binding for 1 h, the resin was washed with 4X 10 mL of 0.5 M NaCl, 10
mM imidazole and 20 mM Tris-HCl, pH 7.5, and the bound protein was eluted with 3X 2
mL of 0.5 M NaCl, 250 mM imidazole and 20 mM Tris-HCl, pH 7.5. The elution fraction
was dialysed into a low salt buffer (50 mM NaCl and 20 mM Tris-HCl, pH 7.5), and
further purified by anion exchange chromatography (MonoQ HR 10/10, AKTA FPLC
System, GE Healthcare) with a linear gradient of 50-500 mM NaCl over 30 min, followed
by a size-exclusion chromatography (HiLoad 16/60 SuperDex-200 column, AKTA FPLC
System, GE Healthcare) with buffer 150 mM NaCl, and 20 mM Tris-HCl, pH 7.5. Pooled
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MdnC was concentrated to ~10 mg mL-1 for crystallization. MdnB was overexpressed
and purified following the same procedure described for MdnC.
Table 4-5. Oligonucleotides for protein cloning and mutagenesis
Protein Primers
MdnB WT 5’-GACCTTCATATGAAAGAATCGCCGAA*
5’-CTAACTCGAGTCAGTGATGGTGATGGTGA
TGGGCGGCGGCACCGAACACCAGAAAATC
MdnC WT 5’-GCCATATGACGGTAGTGATTGTGACG
5’-GACTCGAGTCAGTGATGATGATGATGATG
AGCAGCAGCGGAGTTCACCAGAATCTC
K125A 5’-CGCCAACCACgcaCAACTGCAGC
5’-TGGTCCACTTTGGCAATC
K166A 5’-TATTGTCACTgcAATGCTGTCCCAG
5’-CCGGTCGCTTCAAACTCT
Q207A 5’-GATGACATTTgcAGAAAACATCCCG
5’-GGACAAAATTGCAGACCC
Q215A 5’-AAAGCACTGGcGCTGCGTATTAC
5’-CGGGATGTTTTCTTGAAATGTC
D281A 5’-GGCGCCATTGcTATGATCGTG
5’-ATAGTTCAGGCCGAAATATTTC
E294AN296A 5’-tgcTCCGGTTGGTGAGTTCTTC
5’-atcgCCAGGAAGATATAACGTTCATC
E191KD192K 5’-GGTTACAAAAaaaaaaCTGGATAACCTGG
5’-GGGCTGGTAAAGACGACC
E191AD192A 5’-GTTACAAAAGcagcTCTGGATAACCTGGAG
5’-CGGGCTGGTAAAGACGAC
SeMet M25K 5’- GCGATCGAAGCAAAAGGCAAAAAGGCC
M104L 5’- GTATTCGTGGCCTGATTGCCTCACTGTC
M167L 5’- CGGTATTGTCACTAAACTGCTGTCCCAGTTCG
M181N 5’-GGGGATAAACAGGAGGAAAACGTCGTCTTTAC
M282I 5’-CGCCATTGATATCATCGTGACCCCG *Restriction sites are underlined.
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4.4.2 MdnC Mutagenesis
Mutations of MdnC were carried out using the Q5 Site-Directed Mutagenesis Kit
(NEB) following the manufacturer’s instructions. The validity of mutagenesis was
confirmed by DNA sequencing. The MdnC mutant list and primers designed for the
mutagenesis are listed in Table 4-5.
4.4.3 Designed MdnA Variants
.Chemical synthesized MdnA variants were purchased. Full-length MdnA
(residues1-49) (Genscript, US and SynPeptide, China); MdnA1-35 contains the leader
peptide region only (amino acids 1-35, SynPeptide); while MdnA11-49 (LifeTein, US) and
MdnAAc20-49 (EZBioLabs, US) are shorter versions of MdnA, with truncated amino acids
in the N-terminus leader peptide region.
4.4.4 Macrocyclization of MdnA Variants.
For MdnA (and its variants) cyclization in vitro, MdnA substrates were treated
with 100 mM Tris-HCl, pH 8.0, 2 mM ATP, 10 mM MgCl2, 50 mM KCl, and 1.8 μM
MdnC (or plus 1.8 μM MdnB) at 37°C and quenched at 4 h with equal volumes of 0.5 M
EDTA. HPLC was used for product analysis: 30 % acetonitrile for 3 min, and 30-40 %
acetonitrile from 3-14 min in 0.1 % TFA-water. LC-MS was used to analyze the
corresponding cyclization with a method: 3% 0.5% formic acid (FA)-acetonitrile for 3
min, and 3-98% 0.5% FA-acetonitrile from 5-11 min in 0.5 % FA-water.
4.4.5 MdnC-MdnA Dicyclization Kinetics
The kinetics of MdnC and MdnA dicyclization reaction was analyzed using an
HPLC based assay. Various concentrations of MdnA (8.75 - 52.5 μM) were processed
with 1.8 μM MdnC with the reaction conditions described above at 25°C, and quenched
at various time points (2-60 min). The reaction mixtures were analyzed with analytical
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HPLC equipped with an auto-sampler and the method: 30 % acetonitrile for 5 min, and
30-40 % acetonitrile from 5-11 min in 0.1 % TFA-water. The linear form of MdnA eluted
at 8.5 min, and the MdnC reaction product, dicyclo-MdnA (MdnAΔ2) eluted at 9.5 min.
LC-MS analysis verified that the product was 35.9 Da difference from the standard as
described for microviridin K209, indicate two cyclizations had occurred. Kinetic
parameters were deduced from the integration of the starting material and product
peaks areas. Reactions at each MdnA initial concentration were measured in triplicate.
4.4.6 Leader Peptide Binding Affinity Determination
Isothermal titration calorimetry (ITC) was used to determine the MdnB, MdnC
and MdnA interactions. 200 μL of 0.1 mM enzyme in 50 mM KCl, 10 mM MgCl2 and 100
mM HEPES-NaOH, pH 8.0 was placed in a MicroCal iTC200 reaction cell, while 39.6 μL
full (or partial) length MdnA peptide (1.0 mM) was titrated into the protein solution over
time at 25°C. Heat change during the reaction was detected and recorded for binding
affinity calculation. All titrations were repeated in triplicate. The binding parameters and
corresponding error are calculated using the Origin software package155.
4.4.7 Preparation of SeMet Labelled Protein
Selenomethionine (SeMet) labelled MdnC was produced using methionine
auxotroph E. coli (T7 Crystal Express, NEB). To improve the crystal diffraction quality of
SeMet-MdnC, mutagenesis was carried to decrease the number of non-conserved Met
(M25K, M104L, M167L, M181N, and M282I). Cells were cultured in 6 L M9 minimal
media supplemented with 50 mg L-1 L-Met and grown to an OD600=0.6 at 37°C. The
cells were then pelleted by centrifugation, resuspended in 6 L of fresh M9, and
incubated at 37°C for 3 h to deplete the intercellular methionine. 50 mg L-1 L-SeMet was
then added to the culture 20 min before the overexpression, which was initiated by
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adding 100 μM IPTG followed by growth for 16 h at 18°C. The purification for SeMet
labelled MdnC was the same as described above for the native protein with the addition
of 10 mM dithiothreitol (DTT) to the dialysis, anion exchange, and gel filtration
purification buffers. SeMet labelled MdnB was prepared and purified in the same
fashion.
4.4.8 Crystallization, Data Collection and Crystallographic Analysis
Purified MdnC was mixed with 10 eq. of AMP-PNP, 1.2 eq. of MgCl2 and 1.05
eq. of MdnA1-35 (final concentration [MdnC] = 7.4 mg mL-1) and allowed to equilibrate at
4°C overnight. Initial crystal screening was performed in a vapor diffusion, sitting drop
format using commercial sparse matrix screens. Small plate clusters were identified in a
condition containing 200 mM ammonium sulfate, 20% PEG-3350, and 100 mM bis-Tris-
HCl, pH 6.5. Optimization of salt and pH along with microseeding were performed in
hanging drop format at 20°C.The resultant crystals with a maximum size of
~50×200×200 μm were obtained in a final condition that contained 250 mM ammonium
sulfate, 28% PEG-3,350, 8% dioxane and 100 mM bis-Tris-HCl, pH 5.5.
MdnB was concentrated to 6.0 mg mL-1 and premixed with 5 eq. AMP-PNP and
1.5 eq. MgCl2. The mixture was incubated at 4°C for 4 h and screened with commercial
sparse matrix in a vapor diffusion, sitting drop format. Diffracted plate-shaped single
crystals with a size of ~40×100×100 μm were obtained in the best condition contains
20% PEG 4,000, 20% 2-propanol and 100 mM sodium citrate, pH 5.6. SeMet labelled
MdnB was crystallized in a same manner, crystals were harvested and flash frozen, with
15% glycerol as cryoprotectant.
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Diffraction data for MdnB crystal was collected on beamline 22-ID of the
Advanced Photon Source-Argonne National Laboratory (APS-ANL) at a wavelength of
0.9787 Å. MdnC diffraction data were collected on beamline 21-ID-G of the Life
Sciences Collaborative Access Team (LS-CAT) facility, APS-ANL, at a wavelength of
0.9786 Å. Data were collected at 100 K, integrated, merged and scaled using XDS
package156. The MdnB SeMet-SAD data set was processed into the space group C2
with two molecules per asymmetric unit. Phases were calculated with
PHENIX.AUTOSOL180,159 and the resultant partial model was used in molecular
replacement for native MdnB data. PHENIX.AUTOBUILD succeeded in placing 80% of
the amino acids in MdnB sequence. The remaining residues were built into the electron
density maps manually with COOT160. MdnC crystals diffracted X-ray in space group
P41, with 8 monomers per asymmetric unit. MdnC structure was solved with dimeric
MdnB as molecular replacement model with PHASER157. PHENIX.AUTOBUILD placed
85% of the amino acids in MdnC sequence, while the rest of MdnC as well as MdnA
fragments were built manually with COOT. SeMet labelled MdnC data set facilitated the
phase improvement throughout the model building. Structures were refined using
REFMAC5183 and PHENIX.REFINE159. Sigma-A weighted, simulated annealing
composite omit maps were used to judge and verify structures throughout refinement.
The refined MdnC and MdnB structures include Ramachandran favored (outliers) as
95% (0.6%) and 96% (0.9%), respectively.
Crystallographic data and refinement statistics are shown in Table 4-3. Sequence
alignments were carried with Clustal Omega229. MdnC sequence evolutionary
conservation was calculated from ConSurf230. Protein surface electrostatics were
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calculated and mapped with APBS231 and PDB2PQR232. Structural illustrations were
prepared with Pymol161.
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CHAPTER 5
CRYSTAL STRUCTURES OF Y3 IN FUNGUS Coprinus comatus REVEAL A NOVEL LECTIN FAMILY WITH ANTIVIRAL AND ANTITUMOR ACTIVITIES
5.1 Introduction of Lectins, and Proteinous Natural Product Y3
Lectins are a diverse group of non-immune proteins that exhibit reversible and
specific binding to carbohydrates and have been found in almost all organisms,
including a high prevalence in fungi, plants and animals233. The proteins belong to a
number of unrelated structure classes, with substantially varying carbohydrate
specificities, from monosaccharides to complex oligosaccharides234. Lectin initiated
carbohydrate/protein interaction is a key feature of many fundamental biological
processes and often facilitate in cell adhesion, cell-to-cell interaction, as well as
carbohydrate storage and utilization235. The chemistry and biology of the extraordinary
tight and specific carbohydrates binding lectin is of general interest in terms of its
related biological relevant activities and is a potential target toward the development of
therapeutic treatments. Lectins have been exploited as biomarkers and applied as
diagnostic tools in cancer cell detection, imaging and targeting236,237,238. Additionally, the
potential application of utilizing these carbohydrate-binding proteins as antifungal,
antiviral, or antitumor agents239 is under investigation.
Fungal lectins have attracted significant attention during the past decades as a
source of biomolecules with interesting and novel characteristics. Additionally, lectin
genes account for many uncharacterized ORFs in a variety of species. A number of
novel fungal lectins have been described recently, with a majority arising from
mushroom species240. Mushrooms show great potential for the production of bioactive
metabolites and are a prolific resource for natural product drug leads241,242,243. Most
mushroom lectins are fruiting body cytoplasmic lectins244, with only a few secreted
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mushroom lectins have been reported so far245,246. The biological roles of the mushroom
lectins is proposed to contribute to self-defense against predators244,247. Similar to
lectins from other species, mushroom lectins have significant pharmacological
potentials244, including mitogenic248, anti-proliferative249, antiviral250 and antitumor
activities251. Several mushroom lectins have been structurally identified recently through
X-ray crystallography with diverse folds: BEL in Boletus edulis has a jelly-roll fold252;
CNL in Clitocybe nebularis253 and rMpL in Macrolepiota procera are both β-trefoil folded
lectins254; secreted LDL in Lyophyllum decastes has been reported to form an αβ2α-
sandwich architecture245.
Y3 is a proteinous natural product from mushroom Coprinus comatus and was
previously reported as a probable glycoprotein, and an inhibitor against the
multiplication of tobacco mosaic virus (TMV)255. The amino acid sequence of Y3 was
deposited several years after the initial report (ADK35888.1). A limited number of
homologous sequences, sharing low similarity with Y3, are in current databases and
none has been characterize256 (Figure 5-1A). In order to understand the antiviral (TMV)
mechanisms, as well as the toxicity and pharmacology of Y3, we heterologously
expressed mature Y3 for functional and structural analysis. Purified Y3 contains several
disulfide bridges and interacts with carbohydrates in a non-covalent fashion. The
characterization of Y3 in vitro, along with the high resolution crystal structures indicate
that Y3 belongs to a novel lectin family. Structural identification of Y3 facilitates our
understanding of its antiviral mechanisms, and advances our characterization of its
expended antiviral/antitumor activity. Additionally, Y3 provides a reference for the
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identification of novel lectins and provides a structurally basis for protein-glycan
interactions in situ.
Figure 5-1. Recombinant Y3 expression and characterization. A) Sequence alignment (PRALINE) of Y3 (C. comatus) with uncharacterized proteins from Hebeloma cylindrosporum h7 and Galerina marginata CBS 339.88. Predicted signal peptide regions are in grey; conserved cysteines are in orange; predicted N-glycosylation site (▲) of Y3 is in blue. B) Harvested Y3 from P. pastoris heterogeneous expression was purified with size exclusion chromatography as a probable dimer. Running positions of 8.4-kDa and 42-kDa molecular mass standards are indicated. C) Native and denatured PAGE analysis of Y3, suggested the aggregation of Y3 in solution. D) ESI-MS analysis indicates a mass peak belongs to a single Y3 protomer, excluded the intermolecular disulphate bridge. Calculated mass of Y3 protomer (start from mature peptide sequence •) is 12229.54 Da.
5.2 Characterization of Y3 as a Novel Lectin
5.2.1 Genetic and Biochemical Characterization of Y3
Y3 is isolated from C. comatus, a common fungus often seen growing on lawns,
roads and waste areas, and is an edible mushroom when young257. Full-length Y3
consists of 130-amino acids encoded by a 390-bp gene, with a 12-a.a. N-termini signal
peptide258. Consistent with previous reports as a glycoprotein255, several post-
translational modification prediction servers predict a potential N-glycosylation site
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(Asn92) with a good contrast, as well as additional probable sites of O-
glycosylation259,260. Recombinant mature Y3 was amplified using a heterologous
expression system in P. pastoris for further characterization. The putative signal peptide
of Y3 was excluded in the clone. Meanwhile, to maximally preserve the original
structure and activity, no purification-tag was included.
Figure 5-2. Characterization of purified Y3. A) Phenol-sulfuric acid method was used for total carbohydrates determination. D-glucose (100 ug∙mL-1) or protein (10 mg∙mL-1) were diluted to 400 μL with water, and mixed with 1 mL concentrated sulfuric acid. Purified Y3 contain carbohydrates with w/w ration of 22%. B) LC-MS profile of Y3-containing size exclusive chromatography fractions. Two peaks have been identified in MS (positive mode) trace, only peak II contains proteinous 260 nm UV-vis absorbance. C) Peak II belongs to Y3 protomers with a calculated molecular weight of 12.20 kDa while, D) peak I corresponds to the bound carbohydrates.
Overexpressed Y3 was collected and purified from P. pastoris culture
supernatant. The size exclusion chromatography profile indicates Y3 (MW 12.2-kDa) as
an aggregation form in solution, with a similar retention time as the standard E. coli
maltose binding protein (MBP, MW 42-kDa, Figure 5-1B). Native PAGE analysis
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suggests an even higher level of aggregation. Denaturing SDS-PAGE analysis indicates
a molecular weight of ~14-kDa, consistent with the mass of 12.2-kDa from mass
spectrometry (Figure 5-1CD and Figure 5-2). Based on MS, PNGase-F treatment did
not produce any additional species, indicating the protein is unlikely to be N-
glycosylated. Purified Y3 through heat (90 ºC for 20 min) and acid/base treatment (pHs
3~11, 100 mM pH buffer) retain the same SDS-PAGE profile and still produce protein
crystals in decent size and shape. Ellman's test excluded the presence of free thiols in
the purified protein, suggesting the eight cysteines form four disulfide bridges in mature
Y3. Carbohydrates have been identified in Y3 containing fractions through our
purification. Phenol sulfuric acid analysis indicates a 22% w/w carbohydrates/protein
ratio in the final size exclusion chromatography purified samples (Figure 5-2A). LC-MS
analysis also indicates a series of small peaks as potential carbohydrates fragments.
These peaks do not possess the proteinous UV-vis absorbance at 260 nm (consistent
with protein) and consist of a wide range of molecular weight (Figure 5-2D).
Figure 5-3. tNCS complicates the structural determination of Y3. A) Native Y3 data sets have a non-origin Patterson peak at [0.5 0.0 0.5] with the intensity of 47% of the origin peaks, indicated the presence of the translational non-crystallographic symmetry. B) ARCIMBOLDO ab initio phasing with a library containing three-antiparallel beta strands (library size ~ 8,000). Calculations of rotation functions suggest several top antiparallel β-strand candidates, but none of them was retained in transition function search and packing evaluation.
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Table 5-1. X-ray data collection, processing and structure refinement of Y3.
Data Collection Derivative Form
Small unit cell - 5HA2
Native Form
High resolution - 5HA3
Resolution range (Å) 41.3 - 1.70
(1.76 - 1.70)*
41.8 - 1.18
(1.22 - 1.18)
Diffraction Source 21-ID-F (0.9787 Å) 21-ID-G (0.9787 Å)
Space group P21 P21
a b c (Å) , β (°) 41.1 55.2 41.0, 99.5 53.3 56.1 62.7, 92.7
Total no. of reflections 148850 (11787) 567305 (38773)
Unique reflections 20217 (1790) 113409 (9627)
Multiplicity 7.4 (6.6) 5.0 (4.0)
Completeness 0.99 (0.87) 0.93 (0.80)
<I/σ(I)> 14.57 (3.70) 22.98 (4.44)
Wilson B factor (Å2) 14.0 8.1
Rmerge 0.093 (0.44) 0.041 (0.29)
Rmeas 0.099 (0.48) 0.046 (0.34)
CC1/2 1 (0.93) 1 (0.91)
Structure Refinement
Rwork 0.159 (0.187) 0.172 (0.205)
Rfree 0.194 (0.248) 0.189 (0.240)
No. of non-H atoms 1950 4189
Protein 1751 3420
Ligand 34 52
Protein residues 224 448
RMS Bonds (Å) 0.019 0.006
RMS Angles (°) 1.54 1.05
Ramachandran
Favored (%) 95 97
Outliers (%) 0 0
Clashscore 2.9 1.3
Average B factors (Å2) 18.0 11.9
Protein 16.9 10.0
Ligand 30.5 14.9
Water 27.2 20.7
Pdb entry 5HA2 5HA3 *Values for the outer shell are given in parentheses.
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Figure 5-4. Overall structure of Y3 dimer. A) Y3 dimer forms a 10-stranded β-sheet, helices α1 and α2 locate on one side of the dimeric interface, B) while two α3 locate on the other side. C) Structure topology illustration of Y3 dimer. D) LLD shares a similar protomer structure (pdb entry 4NDV, RMSD of 2.4 Å), but has distinct dimeric packing. E) Plant killer toxin SMKT (pdb enrty 1KVD) with a similar 10-stranded β-sheet forms dimeric heterodimers.
5.2.2 tNCS Complicates the Structure Determination
Native Y3 crystals were harvested in a high pH (pH 9.5) and diffract X-rays to
1.18 Å resolution in space group P21, with unit-cell parameters as a = 53.3, b = 56.1, c =
62.7 Å, β = 92.7º. Integrated data sets possess a large non-origin Patterson peak at
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[0.5, 0, 0.5], with 45.5% of the origin peaks intensity (Figure 5-3A). Matthews’s
coefficient analysis indicates a possible tetramer solution with a low water percentage of
33.6 % (MW 12.4-kDa, Matthews’s coefficient 2.55 Å3∙Da-1). There is no homologous
structure available for using molecular replacement (MR) to solve crystal phases. We
made considerable efforts with ab initio phasing261,262,263 and sulfur/heavy atom (HA)-
SAD phasing, but the calculations failed owing (we believe) to the presence of
translational non-crystallographic symmetry (tNCS). tNCS is described as a pathology
of protein crystals264,265. With multiple copies of a macromolecule found in similar
orientations, phasing and refinement programs commonly confuse crystallographic and
non-crystallographic symmetry, thus produce problematic structure solution. The phase
problem was eventually solved for Y3 by identifying a crystal in with an alternative lattice
(P21, a = 41.1, b = 55.2, c = 41.0 Å, β = 99.5º). This crystal was soaked in K2PtCl4 and
does not exhibit tNCS. The location of HAs was identified with SHELXD188 and the
phase was calculated with SHELXE and refined with additional rounds of MR-SAD266,184
as described70.
5.2.3 Structure of Y3 Reveals a Novel Lectin Family
Y3 consists of two or one dimers (PISA server)130 per asymmetric unit in two
different space groups. Y3 protomer forms a compact single-domain architecture with
an αβα-sandwich, composed of three α-helices and a five-stranded β-sheet. The β-
sheet consisted of strands β1-β4-β5-β3-β2 from edge to central in an antiparallel
orientation (Figure 5-4). Two long α-helices α1 and α2 packed against one side of the β-
sheet, while C-terminus α3 locates on the other side. Helix α3 is a unique figure of Y3
compare with reported αβ2α-sandwich fold lectin (Figure 5-4D). The structure was highly
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ordered, with only short turns and loops connecting the α-helices and β-sheet. As
predicted, there are four intramolecular disulfide bridges further aiding to stabilize the
protomer. Cys6-Cys83 connects β1 and α2, Cys18-Cys51, α1 and β3, Cys43-Cys108,
β2 and α3, while Cys26-Cys72 joins α1 and α2 (Figure 5-5B). The structure clearly
shows that the N-terminus of Y3 is modified267. The α-factor signal sequence was
precluded during the expression, while Gln1 was cyclized and formed PCA1
(pyroglutamic acid, Figure 5-5A) by glutamine cyclase during protein synthesis268. PCA
is the common +1 residue following the signal peptide269. A CHES, buffer molecule from
crystallization condition was well resolved in the electron density maps and located
between β3α2 and β4β5 loops with the anionic sulfate group interacting with Nphe97,
NSer68 and Asn71 sidechain, with the hydrophobic cyclohexane ring extended into the
bulk solvent (Figure 5-5E). The four Y3 protomers (native form) in a crystallographic
asymmetric unit were very similar to one another with an RMSD range of 0.14-0.36 Å.
Protomers differ primarily in β1α1 loop region, with a Cα (backbone carbon) movement
up to 4.5 Å (residues 8-13, Figure 5-5F). The two pairs of five-stranded anti-parallel β-
sheets from two Y3 protomers assembled to large ten-stranded anti-parallel β-sheet,
with all long α-helices (α1 and α2) located on one side of the β-sheet, and short α-
helices (α3) on the other side. In addition, residues 32-35 locate on the α1β2 loop close
to the neighboring β’2 and form intermolecular hydrogen bonds. Residues 106-108 of
α3 also interact with β’2 with electrostatics effect and π-π stacking (Figure 5-5D).
Structural homology searches (DALI server)131 revealed that Y3 shares the
highest structural similarity with an α-galactosyl binding lectin LDL from mushroom L.
decastes, with 12% sequence identity245. LDL is a 96 residues protein consists of a 5-
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stranded anti-parallel β-sheet, two α-helices and three disulfide bridges. The two α-
helices packed against one side of the β-sheet, similar to the long helices α1 and α2 of
Y3 (Figure 5-4D). Y3 is also structurally similar to a plant secreted mannose-binding
lectin Gnk2 (Ginkgo biloba, 7% sequence identity) that exhibits antifungal activity270.
Neither LDL nor Gnk2 shares the same disulfide bridge position compare with Y3. Y3
protomers also form a distinct dimer as compared with LDL or Gnk2. Gnk2 exists as a
monomer in solution271; while LDL forms an alternate dimer by the stacking of the
exposed sides of the β-strands of the two subunits as an αβ2α sandwich272.
Interestingly, SMKT from Millerozyma farinosa273 shares a similar assembly as Y3
dimers. SMKT is a plant acidophilic, antifungal killer toxin and forms dimeric
heterodimers (Figure 5-4E).
5.2.4 Carbohydrates Binding Site
Y3 has a large hydrophilic pocket on the dimeric interface. The pocket sits on the
10-stranded β-sheet plane and is surrounded by two α3 helices, one from each
protomer. Extra electron density was identified in the middle of this pocket near the
sidechains of hydrophilic amino acids Asp102 and Asn104. With the absence of sulfur
anomalous signal, the density is excluded from CHES. Although the exact identity of the
molecular remains unclear, we assumed the molecular to be a carbohydrate fragment.
A molecular of mannose, which is the major carbohydrate unit of P. pastoris was
modeled into the calculated mFo-DFc map (Figure 5-6A). The C2-hydroxyl group is
close to Asp102 and Asn104, stabilized by the hydrogen bonds interaction. Aromatic
residue Tyr7 locates on the bottom of the carbohydrates hexa-cyclic ring and restrains
the molecule in the highly hydrophilic cave.
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Figure 5-5. High-resolution crystal structure of Y3. A) The N-terminus Met0 was cleaved during Y3 expression. PCA1 was formed through Glu1 cyclization. B) Four intramolecular disulfide bridges stabilize Y3 protomers. 2mFo-DFc map is displayed at a 2.0σ contour level. C) HA-derivative Y3 dimer in alternative P21
space group. The anomalous difference map was displayed at a 4.0 σ contour level. Pt3 interacts with a disulfide bridge in an isomorphous fashion. D) Gln41 and Arg’35 stabilized the dimeric Y3. Gln41 and Arg’35. E) A bound CHES molecule was well resolved near β4β5 and β3α2 loops. F) Y3 protomers majorly differ in the β1α1 loop region. The orange loop (chain D) shifted ~ 4.5 Å compare with the loops of other protomers. The α2β4 loop is also noted. G) CCL shares a similar protomer structure, but possesses longer β1α1 and α2β4 loops to assist the protein-carbohydrate interaction. CCL also includes an extra disulfide bridge (*) between β1α1 loop and C-terminus loop.
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The titration of mono- or di- saccharide sugars into Y3 did not produce detectable
heat as measured by ITC, indicative of a binding interaction. The potential interaction
might be blocked by bound polysaccharide glycans upon our purification274. To further
address the protein/carbohydrates recognition, the dominating yeast glycan, poly-
mannose (1’, 4’ linkage) glycans were modeled into the Y3 carbohydrates binding site to
detail the interaction. Hexa-mannose interacted with Y3 dimer, with the 4’-termini
saccharides laid near Asp102/Asn104, and extended across the dimeric interface
through a groove along the flexible β1α1 loop (Figure 5-6B).
Figure 5-6. Y3 interact with carbohydrates in a novel mechanism. A) A mannose molecular was modeled into a hydrophilic pocket near Asp102 and Asn104. Extra electron density in calculated mFo-DFc map in the pocket was displayed at 3.0σ contour level. B) Hydrophobicity map of Y3 dimer. The electrostatic potential (±6 kT∙e-1) is plotted on the solvent-accessible surface. Y3 dimer possesses an amphiphilic ‘Janus’ architecture. Modelled polysaccharide (hexa-mannose) plugged into the middle hydrophilic pocket, and extended through the groove along the β1α1 loop. The 4’-terminus is noted in yellow.
5.2.5 Y3 with Antitumor and Antiviral Activities
Reported Y3 isolated from C. comatus inhibits TMV infection of N. glutinosa
leaves at a concentration of 12.5 μg∙ml-1 protein255. Heterogeneously expressed
recombinant Y3 maintains the reported anti-TMV activity (data not shown). In addition,
we evaluated Y3-mediated tumor cell cytotoxicity. Y3 displayed antitumor activity
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against human cervical cancer (Hela) in a dose-dependent manner, with IC50 value of
13.2 μg∙mL-1. In addition, our preliminary results indicate that Y3 restrict HCV entry into
Huh7.5 cells. The inhibition effect is ~100% at a protein concentration of 200 µg∙mL-1.
5.3 Discussions and Insights
This study presents the functional and structural characterizations of Y3, a novel
lectin from the mushroom C. comatus. Recombinant Y3 is secreted from P. pastoris
harboring the pPICZαA-Y3 plasmid, and shares the same SDS-PAGE profile as well as
the same anti-TMV activity with the C. comatus isolates255. Our purified Y3 contains a
large w/w ratio of carbohydrates as previously reports suggested it is a glycoprotein.
Multiple analyses include mass spectroscopy indicate that Y3 interacts with large
glycans through a non-covalent association. The large aggregation of Y3 in native
PAGE also confirms the hypothesis. Hence, we assigned Y3 as a lectin. The presence
of the signal peptide along with multiple disulfide bridges suggests that Y3 is most likely
secreted in the natural producer. Most mushroom lectins are as cytosolic, with a
majority found in the fruit body, and a few are located in mycelium244. Only a few
examples of extracellular mushroom lectins have been reported so far, exemplified as
LEL from Lentinus edodes246 and LDL from L. decastes245. Suggesting Y3 is a secreted
lectin, the protein remains intact in a broad pH/temperature range, and the extraordinary
high stability expends its pharmacology potential.
Y3 crystallization was carried out using a conventional vapor diffusion method.
Although Y3 crystals diffract X-ray to a high resolution, attempted ab initio phasing did
not provide interpretable electron density maps. tNCS is most likely to be the key
problematic factor. ARCIMBOLDO phasing with a antiparallel β-strand library provided
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rotation function solutions with good contrast, but none of the solutions were retained
during the transition function search and packing evaluation263 (Figure 5-3B). tNCS also
complicated the experimental phasing calculation. The final structural solution was
greatly facilitated by a single ‘anomalous’ crystal with a smaller unit cell and without
apparent tNCS. Of note is the new crystal form has a higher calculated water
percentage (37% compare with 33%), suggesting the new space group might be the
artifact caused by Pt soaking instead of direct dehydration275.
Detailed high-resolution crystal structure does not show protein N/O-
glycosylation, and confirms Y3 as a novel lectin. Dimeric Y3 forms a large bowl-shaped
10-stranded antiparallel β-sheet, with four and two α-helices on either side. Y3
possesses a unique tertiary structure, and does not share a similar folding compare with
other lectins. Reported fungi lectins have been summarized into different structural
classes276, typified by bladed β-propeller fold277, β-trefoil fold278, jelly roll fold273, and
αβ2α-sandwich fold245. Y3 protomer is similar to αβ2α-sandwich protomer. However, with
an additional C-terminal helix, Y3 dimer forms a large αβα-sandwich. This αβα-
sandwich is a novel class of lectin. Several lectins, include L-type lectin279 and C-type
lectin280 requires Ca2+ (and Mn2+) to mediate carbohydrates recognition and interaction.
There is no conclusive metal ion(s) resolved in the native structures, and Y3 may
employ a metal independent carbohydrate interaction. In spite of the fact that Y3
protomer is relatively similar to CCL, Y3 and CCL do not possess a same carbohydrates
interaction mechanism (Figure 5-5FG)245. CCL and Y3 share the same β1α2 disulfide
bridge. The β1α1 loop of CCL has 3 extra amino acids. The longer β1α1 loop, combine
with the longer α2β4 loop (2 amino acid residues longer), provides a large pocket for
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CCL carbohydrates interaction. In addition, CCL contains an extra disulfide bridge
between β1α1LLC loop and C-terminusLLC loop. The extra disulfide bridge partially
restrains the flexibility of β1α1LLC for probable carbohydrate specificity. Partially resolved
carbohydrates bound in a highly hydrophilic pocket of Y3 locates on the dimeric
interface, with the C2’-hydroxyl group interact with Asp102 and Asn104. Tyr7 also help
to stabilize the modeled mannose molecular. Several other lectins have the similar
reported pockets, as the site 1 in CLL also consists of a Glu-Gln-Tyr pocket to interact
with N-acetylglucosamine (GlcNac)281. Other reported systems also use β-stranded
dimeric interface for specific molecular or peptide recognition, exemplified by the
allosteric site of procaspase-3282, 283. In addition, a molecular of CHES is well resolve at
β3α2 and β4β5 loop region of Y3. This region has a critical Asn71 to provide
electrostatics interaction, and shares the similar flexibility compare with the LLC
carbohydrate binding site near the β1α1LLC and α2β4LLC loops (Figure 5-5E)245. The
CHES binding site might also assist the carbohydrates interaction.
Y3 do not show detectable interaction against common mono- or disaccharide
sugars in our ITC based screening. As common with Chlorophyllum molybdites lectin
(CML)274, the Y3 protein might already been non-specifically saturated with longer chain
glycans upon yeast expression/purification, whereas the mono- or disaccharide sugar
usually possess a much low lectin binding affinity284. To better understand the Y3-
glycan interaction, poly-mannose chains (1’, 4’ linkage) in different have been modeled
into Y3 carbohydrate binding pocket with a grid covering the entire Y3 dimer. The
hydrophilic residues near the dimeric interface pocket include Asp102, Asn104 and
Asp111 largely stabilized modeled glycans. The 4-terminus of modeled polysaccharide
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shares a similar protein contact surface compare with the fitted mono-mannose in the
calculated mFo-DFc map (Figure 5-6A). The hexa-mannose chain was extended from 4’
to 1’ along the protein β1α1 loop and interacts with several additional key residues as
Asp8 and Asn9, while 4’-terminus could also extend with the same fashion (Figure 5-
6B). Further determination of Y3-glycan specificity is still under investigation. However,
from the calculated protein contact potential map285, Y3 dimer forms a ‘Janus’
conformation, with all hydrophilic residues assembled in the on side, while the other
side is almost completely hydrophilic (Figure 5-6B). This is also a unique feature
compare with other reported lectins, and extend the pharmacology potential of Y3 in
preventing specific intercellular contact. Y3 may perform as ‘molecular lid’ to cover the
glycan chains on the cell /virus surface, and create a hydrophobic shell to prohibit the
cell (virus)-cell interaction. Structural information provides valuable insight into the
potential antiviral and antitumor activity of Y3.
With the information above, the anti-TMV mechanism of Y3 can be predicted.
TMV is archetype positive-sense single stranded RNA virus. Although the monomeric
unit TMV coat protein of does not possess glycosylation, reported lectin AAL (from
Agrocybe aegerita) interact with TMV in isoelectric focusing (IEF) in vitro250. In addition,
Y3 may interact with specific receptor /transporter on plant leaves and competes with
TMV binding and entry. Y3 could employ a similar antiviral mechanism as PR-1
(pathogenesis-related protein-1). RP-1 gene encodes an 168-amino acid extracellular
protein with an αβα sandwich architecture, and reacting hypersensitively to TMV286,287.
Pathogenesis-related (PR) genes, as well as the directly the protein secretory pathway
genes have been regulated by NPR1, the key factor relates to systemic immune
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response in plants288. However, the substrate for PR-1 is unknown yet. Y3 is also a
potential candidate for TMV-inhibition in vivo. Transgenic plant N. tabacum with
overexpressed BanLec-1 (lectin from Musa paradisiacal) has an inhibited local spread
level of TMV compares to WT289.
In addition, we employed a broad antiviral and antitumor screen for Y3. Detected
antitumor activity of Y3 against human cervical cancer (Hela) has an IC50 value of 13.2
μg∙mL-1 (Figure 5-7A). Intriguingly, Y3 almost completely restrict HCV entry into Huh7.5
cells at a protein concentration of 200 µg∙mL-1 (Figure 5-7B). We predict that the activity
and specificity of Y3 can be further tuned by altering the amino acid residues on the
protein hydrophilic surface (Figure 5-6B). Designed mutagenesis studied and
corresponding activity assays are currently ongoing.
In sum, lectins employ widely different strategies to generate ligand specificity
and multivalency. The low primary sequence identity and diverse structural features
often complicate the identification and characterization of novel lectins290. As a novel
family of lectin, Y3 possess unique structures as well as unique biomedicinal potentials.
Our structural insight allowed us in understanding the mechanism of its specific
activities, and engineering the protein function rationally.
5.4 Materials and Methods
5.4.1 Heterologous Expression of Mature Y3
A codon-optimized gene encoding the leader peptide free Y3 (Eurofins
Genomics) was PCR amplified with primers Y3FXh (5’-GTATCT CTCGAG
AAAAGACAAGATCCTTTG) and Y3RNt (5’-TTTTCCTTTTGCGGCCGC
TTAAAAATCAGTGG), digested with XhoI and NotI, and ligated into pPICZαA plasmid.
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Circular pPICZαA-Y3 was linearized with SacI endonucleases and transformed into P.
pastoris X-33 following the manufacturer’s instructions (Pichia Easycomp, Invitrogen) for
expression. Cultures (500 mL YPD) were grown at 30ºC for 48 h to an OD600 12~18 and
harvested by centrifugation (2,000g, 10 min). Cells were resuspended in 150 mL of
BMMY media (100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4×10-5 biotin, and
0.5% v/v methanol) for protein overexpression. Methanol was added every 24 h to a
final concentration of 0.5% to maintain cell induction. Cell culture supernatant was
harvested on day 3 and dialyzed against PBS buffer (pH 7.4) for 48 h. The supernatant
was further concentrated (10,000 MWCO, Millipore centricon) and purified with gel
filtration chromatography (HiLoad 16/60 SuperDex-75 column, AKTA FPLC System, GE
Healthcare) with running buffer, 150 mM NaCl, and 20 mM Tris-HCl, pH 7.5. Purified Y3
was concentrated to 10.0 mg∙mL-1 (600 μL) for further analysis and crystallization.
Protein concentration was determined with Bradford assay (BSA as standard).
5.4.2 Ellman's Test for Free Thiol Determination
Standard Ellman's test was used to detect the free thiol groups in purified Y3
protein291. 1.0 μL Y3 (10 mg∙mL-1) was mixed with 1 mL DTNB reagent (GoldBio) and
incubate at room temperature for 5 min. The optical absorbance at 412 nm was
measured and recorded.
5.4.3 Carbohydrate Content Determination
Concentrated Y3 containing fractions from size exclusion chromatography was
processed with phenol-sulfuric acid method for total carbohydrates determination292.
Protein solutions (0.2-10 μL, 10 mg∙mL-1) were diluted to 400 μL with water and mixed
with 10 μL of 80% w/w phenol. 1.0 mL sulphuric acid was quickly added into the mixture
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and stand for 10 min before 10 min water bath at 25ºC. UV-vis absorbance was
recorded at 490 nm. Protein free blank and D-glucose standards were also prepared.
5.4.4 Carbohydrates Specificity Determination with ITC
Isothermal titration calorimetry (ITC) was used to analyze carbohydrate
specificity of Y3. Purified Y3 was diluted to 1.2 mg∙mL-1 (100 μM) and dialysed against
150 mM NaCl and 20 mM Tris-HCl, pH 7.5 for 16 h. 200 μL protein was placed into a
MicroCal iTC200 (Malvern) reaction cell, while different oligosaccharides in same buffer
at a concentration of 1-5 mM were injected into the reaction cell over time at 25°C. D-
Glucose, α-D-mannose, β-D-galactose and lactose were tested with ITC. Raw data were
integrated, normalized and evaluated using ORIGIN 7.0 according to the one-site
binding model.
5.4.5 Protein Mass Analysis
For ESI mass spectrometry analysis, protein crystals were picked, washed with a
solution in crystallization condition, and dissolved in 3.0 μL water. MALDI-TOF-MS used
sinapic acid as matrix (Protea). Protein samples treated using a standard PNGase-F
digestion protocol (New England Biolabs) and LC-MS (Agilent) was used to the bound
carbohydrates. Protein fractions harvested from size exclusion chromatography was
analysis with a method: 3.0 % - 60 % of 0.1 % TFA-acetonitrile in 0.1 % TFA-water over
40 min with a flow rate of 1.0 mL∙min-1 (ZORBAX SB-C18, Agilent).
5.4.6 Crystallization of Y3
Initial Y3 crystallization was carried in a vapor diffusion sitting drop format with a
homemade matrix screen designed for glycoproteins (CrystalQuick 96 well sitting drop
plate). The homemade matrix screen adapted common conditions reported for other
glycoproteins. Comparison with commercially-available matrix screens, the homemade
141
matrix contains a broader pH range (pH 3.0 ~ 11.5) and a lower precipitant (PEGs)
percentage. Small plate clusters of Y3 crystals were identified in a condition containing
20% PEG-4000 and 0.1 M CHES, pH 10.5 in 15 min at 25ºC. Optimization of precipitant
and pH, along with microseeding were performed in a hanging drop format (24 well VDX
crystallization plate). Protein (1.8 μL, 10 mg∙mL-1) plus 2.0 μL of precipitant were
balanced against 1 mL of reservoir solution. The resultant plate-shaped single crystals
with a size of 20×20×100 μm were obtained in a final condition that contained 16%
PEG-4000, 10% v/v glycerol and 0.1 M CHES, pH 9.5 in 48 h. Crystals of suitable size
were harvested and flash frozen in liquid nitrogen with an additional 10% glycerol as
cryoprotectant. Heavy atoms derivative crystals for single wavelength derivative (SAD)
and multiple isomorphous replacement (MIR) were obtained through co-crystallization
or soaking. Compounds (Heavy Atom Screen Kits) contain derivative elements (Pt, Au,
Hg, I) were selected according to their solubility (pH 9.5~10.5). Cocrystallization and
soaking procedures were detailed (Table 5-2).
5.4.7 Diffraction Data Collection and Processing
Y3 native and derivative crystal X-ray diffraction data sets were collected on
beamlines 21-ID-G and 21-ID-F of the Life Sciences Collaborative Access Team (LS-
CAT) facility at the Advanced Photon Source (APS), Argonne National Laboratory (ANL)
with a wavelength of 0.9786 Å at 100 K. Sulfur-SAD or Iodine derivative data sets were
collected on in-house X-ray facility (Cu Kalpha source, R-AXIS IV++ imaging, Rigaku)
with a wavelength of 1.5418 Å at 100 K. Data sets were merged using XDS program
package156 and then scaled with AIMLESS from the CCP4 suite182 to space group P21,
in two diverse forms with different unit cell parameters. Y3 native crystals diffract X-ray
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to 1.18 Å, while the Pt-derivative data set was truncated at 1.70 Å resolution (Table 5-
1). Native data sets in have large non-origin Patterson peaks as indicated by
PHENIX.XTRIAGE from PHENIX suite159, suggested the presence of translational
noncrystallographic symmetry (tNCS)264.
Table 5-2. Heavy atom derivative crystal preparation of Y3
Concentration
Co
Cry.
Soaking Time
30 s 10 m 4 h 24 h
Potassium iodide 500 mM -* - ׆ ×
5-amino-2,4,6-triiodoisophthalic acid 3 mM - - - ×
Potassium tetrachloroplatinate(II) 10 mM
-‡ + +#
Potassium tetracyanoplatinate(II) 10 mM
+ ×
Gold(I) potassium cyanide 10 mM
- + +
Gold(III) chloride 10 mM
- +
Sodium tetrachloroaurate(III) 10 mM × × ×
Mercury(II) chloride Satu.
- -
Mercury(II) nitrate monohydrate Satu.
- -
Methyl mercury Satu.
- -
Ethyl mercuric phosphate Satu.
+ +
Ethyl mercury chloride Satu.
+ + *
Crystals without detectable anomalous signal; †crystals unable to harvest or do not diffract;
‡crystals with
detectable anomalous signal; and #protein crystals harvested in the alternative space group.
5.4.8 Atomic Structure Determination and Refinement
The structure of Y3 was determined with Pt-SAD. The number and location of
heavy atoms were identified with SHELXD (Figure 5-5C)188. Initial phase calculation
was carried using SHELXE188, and further refined with MLPHARE185, PIRATE186 and
PHASER157. An interpretable electron-density map was generated with SOLOMON with
20% solvent flattering293. The model building was initiated with SHELXE, and completed
using BUCCANEER181, ARP/wARP189 and COOT160. Final coordinates were refined
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with REFMAC5183 and PHENIX.REFINE158. Ligands were added manually with
COOT160 upon careful inspection of the electron-density maps. Derivative ions were
added manually into the calculated anomalous difference map, with their occupancy
and anisotropic B-factors refined sequentially. The quality of the models were evaluated
using a sigma-weighted, simulated annealing composite omit map. Structural
illustrations were prepared with PyMOL161. Statistics on data collection and atomic
structure refinement are given in Table 5-1. The refined coordinates have been
deposited in the protein data bank (accession codes 5HA2 and 5HA3).
5.4.9 Models of Oligosaccharides Bound Y3
Fitting of oligosaccharides into the Y3 dimeric interface was conducted with
AutoDock 4.2191. Poly-mannose (1’, 4’ linkage, n = 3~6) were constructed with
eLBOW294 and further energy minimized with Spartan ’08192. Standard algorithms and
docking procedures were used for a rigid protein and a flexible ligand in a grid covering
the entire protein dimer. The best docking poses were analysed with AutoDockTools.
5.4.10 MTT Assay
The MTT assay was performed using human cervical cancer (Hela) cells. The
cells were cultured in DMEM medium containing 10 % fetal bovine serum and 100
U∙mL-1 penicillin and streptomycin, and maintained at 37°C in a humidified incubator
under 5 % CO2. The cells (104 cells in 100 μL) were seeded onto 96-well microtiter plate
and incubated overnight. Purified Y3 (final concentrations: 0, 5, 10, 20, 40, 80, 160
μg∙mL-1) were added into the wells. After incubation at 37 °C for 72 h, 10 µL of MTT (5
mg∙mL-1) in PBS was added and incubated for 4 h, followed by the aspiration of the
medium. Dimethyl sulfoxide (DMSO, 100 μL) was added to each well to dissolve the
MTT in the wells and the plate were agitated for 1 h. The optical density (OD) was
144
measured at 570 nm. The IC50 values for Y3 treatment of the tested tumor cells indicate
the Y3 concentrations causing 50 % cell inhibition (SPSS).
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CHAPTER 6
HOMOCYSTEINE METHYLTRANSFERASE MmuM FROM Escherichia coli FACILITATES L-METHIONINE BIOSYNTHESIS AND DAMAGED COFACTOR (R,S)-
S-ADENOSYL-L-METHIONINE REPAIR1
6.1 Metabolite Damage is an Under-Recognized Fact of Life
Metabolites are usually restricted to small molecule natural products that are the
intermediates and products of metabolism295,296,297. Metabolites are recognized and
acted upon by enzymes, act as regulators that control the pace of metabolism, and
serve important biological functions in the cells. While cells have a well-developed
machinery of metabolites synthesis, utilization and degradation, many metabolites are
found damaged in vivo by spontaneous or enzymatic side-reactions298,299. This
metabolite damage occurs on a large scale in all organisms and produces metabolites-
like side products, which is useless and often toxic300,301. Thus, the damage-control
systems are established either to restore damaged metabolites to their original state or
to convert dangerous compounds harmless299,302. Metabolite damage and its control are
analogous to DNA and protein damage and repair, but have been far less studied and
remain poorly understood303,304.
6.2 Homocysteine S-Methyltransferase and Metabolite Repair
L-Methionine (Met) is a structurally unique amino acid with a hydrophobic
thioether side chain305. Although Met is one of the less abundant proteinogenic amino
acids, it has an indispensable role in the initiation of translation306 and commonly
contributes to the hydrophobic cores of proteins307. Met is also the main cellular carrier
of methyl groups as a key component of S-adenosyl-L-methionine (AdoMet), the
1 Adapted with permission from Li, K., Li, G., Bradbury, L. M. T., Hanson, A. D. & Bruner, S. D. Crystal
structure of the homocysteine methyltransferase MmuM from Escherichia coli. Biochem. J. 473, 277–284
(2016) DOI: 10.1042/BJ20150980. Copyright © 2015 The Author(s)
146
universal methyl donor308. Met biosynthesis in bacteria, fungi, and plants is well
understood309,310. The biosynthetic pathway proceeds from aspartate via homoserine
and cystathionine to homocysteine (Hcy), whose thiol group is then methylated to give
Met311. Met is converted to AdoMet in a reaction mediated by S-adenosylmethionine
synthetases, EC 2.5.1.6 (also known as methionine adenosyltransferases).
Figure 6-1. HMT converts L-homocysteine (Hcy) to L-methionine (Met) with unique substrate selectivity. A) HMT (MmuM)-catalyzed methionine biosynthesis with SMM or (R,S)-AdoMet, while structural analogs, B) (S,S)-AdoMet and, C) S-ribosylMet are not active substrates for MmuM.
Plants have a unique additional reaction in which Met is S-methylated to yield S-
methyl-L-methionine (SMM) by methionine S-methyltransferase (MMT, EC 2.1.1.12)312.
SMM can serve as a methyl donor to Hcy in a reaction mediated by homocysteine S-
methyltransferase (HMT, EC 2.1.1.10), an enzyme present in plants, bacteria, fungi,
and animals. The presence of both MMT and HMT in plants sets up a cycle (the SMM
cycle) that allows SMM to serve as a storage and transport form of Met313,314,315. The
occurrence of HMTs in bacteria, fungi, and animals311,316,317,303 enables the use of plant
SMM as a source of Met303,318. Additionally, almost all HMTs can also use the non-
natural (R,S) form of AdoMet, which is generated in vivo by spontaneous racemization
of the natural (S,S) form319 and which cannot serve as methyl donor for other
147
methyltransferases316,317. The ability to process (R,S)-AdoMet enables HMTs to recycle
it back to S-adenosylhomocysteine (AdoHcy) and re-enter the methyl cycle316,317,299.
HMT can thus serve as a repair enzyme for damaged AdoMet, and this is probably its
ancestral function, the ability to use SMM as methyl donor having arisen later in
evolution303. An additional evolutionary innovation is the capacity of HMT from the
selenium-resistant plant Astragalus bisulcatus to use selenocysteine as preferred
methyl acceptor320.
Escherichia coli K12 strain MG1655 has an HMT; this enzyme is encoded by the
mmuM gene, which is in a two-gene operon with the SMM transporter gene mmuP 321.
E. coli MmuM is a 310-residue protein that, like most other HMTs, can use either SMM
or the ‘damaged’ (R,S)-AdoMet as methyl donor; it shows a moderate preference for
SMM and cannot use (S,S)-AdoMet303. Surprisingly, E. coli MmuM - and most other
HMTs - cannot use S-ribosylmethionine (S-ribosylMet) as a methyl donor although this
compound is structurally intermediate between SMM and (R,S)-AdoMet (Figure 6-1)303.
MmuM and other HMTs show sequence similarity to Zn-binding enzymes:
betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), which uses betaine as
the methyl donor322, and methionine synthase (MetS, EC 2.1.1.13), which uses 5-
methyltetrahydrofolate (5-MTHF) as the methyl donor323. Structural prediction indicates
that HMTs contain a functional Zn-binding domain. Although several BHMT and MetS
structures have been reported, no structural information is available for any HMT
protein. In this chapter, I present the first HMT protein structure, MmuM from E. coli.
Structures in the oxidized (Cys disulfide), apo (Zn2+-free), and metallated (Zn2+-Hcy)
forms provide a comprehensive view of the active site pocket and allows insights into
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substrate selectivity, substrate binding, and the methyl transfer mechanism for the HMT
family. The project is collaboration between the Bruner group and the Hanson group
(Prof. Andrew D. Hanson, Horticultural Science Department, University of Florida).
6.3 HMT MmuM in E. coli
6.3.1 Biological Functions and Distribution of bacteria HMT
MmuM enables certain E. coli strains324 to utilize the plant compound SMM as a
methyl donor in methionine biosynthesis. Most E. coli strains also possess two
additional methionine synthases, MetE and MetH that catalyze the S-methylation of Hcy
using 5-MTHF as methyl donor. MetE is an 85-kDa monomer with a Zn cofactor325 while
MetH is a 136-kDa monomer containing Zn and cobalamin cofactors326. The E. coli
KL19 ΔmetE ΔmetH strain but not the ΔmetE ΔmetH ΔmmuM strain can utilize SMM as
a Met source321, indicating that SMM is a biologically relevant methyl donor.
Additionally, the ΔmmuM ΔmetK mutant in E. coli K-12 ilvA pSAMT (carrying the
AdoMet transporter gene from Rickettsia prowazekii) requires more Met for cell growth
than the ΔmetK (AdoMet synthetase) single mutant grown in the presence of equal
AdoMet327. This observation suggests that AdoMet is a methyl donor for MmuM in vivo.
Besides E. coli, the mmuM gene is present in several clinical pathogens, in most cases
encoded on the chromosome. Several mmuM genes are, however, found encoded on
plasmids, increasing the possibility of horizontal transfer and dissemination of mmuM in
bacteria. Interestingly, humans and other mammals also have a MmuM-like enzyme
with specific activity against SMM, and to a much lesser extent, (S,S)-AdoMet328. This
enzyme (hBHMT2) is annotated as a member of the BHMT family, not the HMT family,
although it does not use betaine as methyl donor329,330.
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Table 6-1. X-ray data collection, processing and structure refinement of MmuM
Data Collection Oxidized form
Disulfide bridge
Apo form
Zn2+ free
Metallated form
Zn2+ and Hcy
Resolution (Å) 34.39 - 2.44 (2.54 - 2.44)*
38.42 - 2.89 (2.99 - 2.89)
24.23 - 1.78 (1.84 - 1.78)
Space group I222 P21212 I222
a, b, c (Å) 78.45 84.46 85.82
85.82 85.94 79.02
78.95 85.92 87.66
Total no. of reflections
58379 (6367) 82600 (7945) 141424 (13523)
Unique reflections 10331 (1158) 13530 (1302) 27033 (2711)
Multiplicity 5.7 (5.5) 6.1 (6.1) 5.2 (5.0)
Completeness (%) 99.76 (97.69) 99.71 (98.41) 93.40 (95.36)
<I/σ(I)> 15.8 (3.4) 11.9 (3.5) 14.8 (2.5)
Wilson B factor (Å2) 40.7 41.6 18.1
Rmerge 0.082 (0.49) 0.141 (0.68) 0.093 (0.60)
Rmeas 0.090 (0.59) 0.161 (0.70) 0.104 (0.72)
CC1/2 0.999 (0.996) 0.996 (0.932) 0.996 (0.735)
Structure Refinement
Rwork 0.202 (0.276) 0.225 (0.280) 0.181 (0.257)
Rfree 0.217 (0.296) 0.270 (0.324) 0.206 (0.282)
No. of non-H atoms
Protein 2169 3932 2190
Ligand - 10 13
Water 32 13 179
RMS Bonds (Å) 0.009 0.005 0.007
RMS Angles (°) 1.26 0.92 1.12
Ramachandran
Favored (%) 95.49 96.00 98.00
Outliers (%) 0 0.2 0.35
Clashscore 7.69 3.81 2.05
Average B factors
Protein 47.2 42.7 23.3
Ligand - 39.3 37.0
Water 43.2 38.8 35.7
pdb entry 5DML 5DMN 5DMM *Values for the outer shell are given in parentheses
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Figure 6-2. Overall structure of E. coli MmuM. A) Side and, B) top view of overall structure of MmuM in the form of a (α/β)8 TIM barrel; C) topological diagram of the MmuM monomer. Missing loop (260-275) is boxed in cyan. D) Size exclusion chromatography of purified MmuM, suggesting that the protein is monomeric in solution. Running positions of 66-kDa and 29-kDa molecular mass standards are indicated.
6.3.2 Overall Structure of MmuM in E. coli
Purified recombinant MmuM has a molecular mass of approximately 35-kDa as
estimated on SDS-PAGE, and a calculated molecular weight of 34.49-kDa. The protein
crystallized in the space group I222 or P21212 and the structures were determined using
the deposited structure of T. maritima MetS (PDB entry 1Q7M, N-terminal domain) as
an initial molecular replacement search model. MmuM crystals were identified in the
oxidized (cysteine disulfide), apo (Zn2+ free) or metallated (Zn2+-Hcy) forms. The final
coordinates were refined to between 1.8~2.9 Å resolution with one or two monomers of
151
MmuM present in the asymmetric unit (Figure 6-2 and Table 6-1). Crystallographic
packing analysis using the PISA server130, along with the size exclusion
chromatography elution profile (Figure 6-2D), are both consistent with MmuM being a
monomer in solution. The resolved metallated and oxidized structures in the space
group I222 contain ordered residues 4-259 and 275-310. The missing residues (260-
274) constitute a loop region between β7 and α7 are presumed disordered. The apo
form, P21212, crystals have three additional disordered regions (34-39, 72-78 and 127-
138, Figure 6- Figure 6-3A). The overall crystallographic packing between I222 and
P21212 is similar. MmuM monomers have electrostatic interactions between α2’/α1 and
the neighbouring α4 region. Relative to the I222 structures, the second monomer in
space group P21212 has the region containing α1, α2 and α8 shifted ~ 3.0 Å relative to
other monomers (Figure 6-3B).
Figure 6-3. MmuM apo (P21212) and metallated (I222) form crystals have different crystallographic packing. A) Three missing loops (dark blue, superimposed from metallated form) in P21212 monomer A (grey) locate on the protein top surface. B) MmuM packs in a different fashion in different space groups. P21212 monomer B (light blue) was aligned with I222 monomer (dark blue), while P21212 monomer A (grey) shifted ~ 3 Å against I222 crystallographic symmetry copy (orange).
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Figure 6-4. MmuM belongs to the HMT superfamily of proteins. Sequence alignments (Clustal W2) of MmuM from E. coli with HMTs from Bacillus subtilis (BSHMT) and Arabidopsis thaliana (ATHMT1), with MetS from T. maritima (MetH-Tm), and with BHMT from rat liver (rBHMT). Zn2+-binding cysteine residues are in red. Critical residues (Y71 and T169) for MmuM activity are in green. HMT unique α’5/ α’6 loop region is in blue.
MmuM forms a globular compact structure composed of an (α/β)8 TIM barrel331,
with eight outside α-helices and eight inside parallel β-strands that alternate along the
peptide backbone (Figure 6-2). The protein sequence starts from the α’1 helix, located
at the base of the (α/β) barrel. A missing loop (260-274) is located on the outer surface
of the protein, opposite the metal/substrate-binding site. Two short helixes α’2/α’3
between β1 and α1 are located on the top of the (α/β) barrel covering the β1, β2 and α1
region, while α’5/α’6 (inserted between β3/α3) cover the top of β3 - β5.
Structural homology searches (DALI server)131 reveal that MmuM shares the
highest structural similarity (Z-score of 33.6) with the Hcy-binding domain of T. maritima
MetS (MetH-Tm, PDB entry 1Q7Z)323. MetH-Tm contains two domains, the N-terminal
153
Hcy binding domain and the C-terminal 5-MTHF-binding domain, linked by a flexible
loop of ~12 amino acids. The RMSD value is 2.0 Å for 263 aligned Cα atoms between
the N-terminal domain of the MetH-Tm and the MmuM monomer. MmuM also shares a
high structural similarity with BMHT from rat liver (rBHMT, Z-score of 27.7, PDB entry
1UMY) with an RMSD value of 2.4 Å for 261 aligned Cα atoms between the N-terminal
domain of the rBHMT and MmuM monomers. rBHMT contains an extended ~30-residue
C-terminal region, which ends as a long α-helix that is involved in assembly of the active
tetramer322,332. HMT has an elongated α2 and an additional helix-turn-helix at α’5 and
α’6; while BHMT and MetS have a much shorter β3-α3 loop (Figure 6-4). All three of
these proteins have a conserved Cys on β6 and a Cys-Cys group at β8. These three
cysteines are metal-binding and form the enzyme active site.
6.3.3 The Zn2+-binding, Active Site
MmuM requires Zn2+ for enzymatic activity303. The oxidized MmuM structure is in
a metal-free state with a disulfide bridge between Cys229 and Cys295 (Figure 6-5A).
The biological role of oxidized form MmuM is not known, however the oxidized protein
can be reversibly reduced and charged with zinc to generate the metallated form. The
MmuM apo form (also Zn2+ free) has Cys229 and Cys295 in the reduced thiol form with
a sulfur-to-sulfur distance of 4.3 Å (Figure 6-5B). This suggests the observed apo form
is in an intermediate state between the oxidized and metallated forms. In contrast, the
metallated form has Cys229, Cys295 and Cys296 coordinated to a Zn2+ center with
distances of 2.4, 2.1 and 3.2 Å, respectively (Figure 6-5C). The coordination of Hcy to
metal with a distance of 2.8 Å contributes to a near-ideal Zn2+-centered tetrahedron
geometry. Additionally, Cys296 in the metallated form appears to form a (partial
154
occupancy) disulfide bond with a molecule of β-mercaptoethanol, BME (Figure 6-5C).
BME was introduced into the system during the protein dialysis step incorporating Zn2+
into the protein. The oxidized and metallated forms are similar, with an RMSD of 0.28 Å.
In contrast, the crystals of apo MmuM are in a different space group, with a RMSD of
0.42 Å as compared to the metallated form. With the absence of the Zn2+ center, or the
disulfide covalent linkage, several disordered loop regions are located on the top of the
TIM barrel, including the loop region between β2 and α2 surrounding the active site
pocket. This observation suggests that the Zn2+ center plays an important structural role
in addition to being a key player in catalysis.
Figure 6-5. The Zn2+ binding active site of MmuM. The structure of MmuM showing the Zn2+ binding pocket in, A) oxidized, B) apo and, C) metallated forms; D) Zn2+ binding site in rBHMT with thiolate ligands and Tyr160, critical for enzyme activity; and (E) MetH-Tm with bound Cd2+ (mimicking Zn2+) and substrate Hcy. 2mFo-DFc maps were shown at a contour level of 1.5σ.
155
Table 6-2. Kinetic parameters for HMT MmuM. MmuM activity was measured as described in the Experimental Section. Affinities for the substrates in mutants with less than 5% of the WT activity were not determined (n.d.). Standard deviation was calculated from three independent titrations
Relative Vmax (%) Km (μM, SMM)
WT 100 ± 2.6 45.2 ± 2.4
WT (no K+) 101.4 ± 4.2 73.8 ± 6.8
Y71F 8.7 ± 3.6 127.5 ± 55.7
Y71A n.d. n.d.
Y71T n.d. n.d.
Y71FT169Y n.d. n.d.
Figure 6-6. MmuM catalyzed kenetics. A) ITC-based MmuM (WT)-catalyzed methyl transfer reactions with SMM as co-substrate and B) converted thermal power (dq/dt) ∙ [SMM] plot.
Compared with other similar structures, MmuM has a unique Zn2+-binding pocket.
In the structure of rBMHT, the Zn2+ center is coordinated with three cysteine residues
and Tyr160 on β4 as the fourth ligand (Figure 6-5D). A tyrosine at this position is a
hallmark of the BHMT family. Mutagenesis studies have confirmed the key role of this
tyrosine, as none of the mutants (Tyr160Ala, Tyr160Phe or Tyr160Thr) has significant
BHMT activity332. HMT, as well as MetS family proteins, have a conserved threonine
(Thr169) instead of tyrosine at the corresponding position on β4. MmuM does have a
156
tyrosine (Tyr71) close to the Zn2+ binding site from β2. Tyr71 is highly conserved in the
HMT family, but is replaced by phenylalanine in the MetS family of proteins.
Figure 6-7. Stability of MmuM mutants. CD spectra of WT and mutant MmuM are shown. Spectra are recorded at a wavelength range of 200 - 250 nm
The co-substrate Hcy binds the Zn2+ center at a similar position in MmuM and
MetH-Tm (Figure 6-5E), and is sandwiched between a conserved Phe (MetH-Tm) or Tyr
(MmuM) and Zn2+. To probe the role and function of the Tyr71 near the metal binding
site, sets of site-directed mutants were designed and assayed. Y71F mutant MmuM has
decreased activity against co-substrate SMM, but retains a Vmax ~10% of that measured
of the wild type form (Figure 6-6, Table 6-2). Meanwhile, the Y71A mutant is > 20-fold
less active compared with wild type. This suggests that loss of the large aromatic side
chain significantly interferes with substrate recognition and interaction. Interestingly, the
Y71T mutant has no detectable activity. As all the MmuM mutants retain similar
secondary structure elements as WT protein (Figure 6-7), a possible explanation is that
the threonine hydroxyl group provides a hydrogen bonding interaction, forcing the
substrate Hcy away from its active conformation. To gain further insight into the
differences between the HMT and BHMT active site arrangements, a double mutant
containing Y71F and T169Y was designed to better mimic the BHMT substrate binding
157
pocket; this mutant displayed no activity, however. These data indicate that Tyr169 in
MmuM T169Y mutant may interact with Zn2+ in much the same way as Tyr160 does in
rBHMT (see Figure 6-5D).
Figure 6-8. MmuM substrate binding and recognition. Different methyl donors were modeled with metallated form protein. Bound Hcy (green) locates on the top of the pocket. A) Modeled (R,S)-AdoMet probed near Hcy; B) detailed interactions between (R,S)-AdoMet and Hcy. The S-methyl group in (R,S)-AdoMet is close to the Hcy thiol group, facilitating the methyl transfer. C) Modeled (S,S)-AdoMet; D) (S,S)-S-ribosylMet; E) (R,S)-S-ribosylMet and, F) SMM near active site respectively.
6.3.4 Modeling MmuM Substrate Recognition and Binding
The catalytic mechanism of BHMT has been dissected before, demonstrating
that the reaction may proceed through a transition state where the activated methyl
group reacts directly with the homocysteine thiol group333. HMT is both structurally and
functionally similar to BHMT, and likely retains a similar transition state of the methyl
158
transfer chemistry. The structure of metallated MmuM has the co-substrate Hcy bound
to the Zn2+ center, but the position of the other co-substrate, the methyl donor, was not
apparent from any of our attempts at co-crystallization. The various enzymes that
methylate Hcy use diverse methyl donors: MetS uses 5-MTHF, and BHMT betaine,
whereas the HMT family proteins generally utilize SMM, (R,S)-AdoMet, or both.
Previously, we demonstrated that MmuM can use SMM or (R,S)-AdoMet as the methyl
donor, but not (S,S)-AdoMet or S-ribosylMet303. To understand the basis for the
observed substrate preference, and the reaction mechanism, co-substrates SMM and
(R,S)-AdoMet, and substrate mimics (S,S)-AdoMet and S-ribosylMet were docked into
the metallated form of MmuM to detail the protein /substrate interaction (Table 6-3 and
Figure 6-8).
Table 6-3. Docking statistics of metallated MmuM (with Hcy) and different methyl donors
Methyl Donor No.
Cluster Rank 1 Cluster Rank 2
Free Energy of Binding†
Binding Constant‡
Free Energy of Binding
Binding Constant
(R,S)-AdoMet 300 -7.79 kcal mol-1 1.95 μM -6.21 kcal mol-1 27.9 μM
(S,S)-AdoMet 300 -7.09 kcal mol-1 6.39 μM -6.05 kcal mol-1 36.6 μM
(S,S)-ribosylMet 200 -4.21 kcal mol-1 827 μM -3.19 kcal mol-1 4.61 mM
(R,S)-ribosylMet 200 -4.13 kcal mol-1 931 μM -3.98 kcal mol-1 1.21 mM
SMM 200 -6.42 kcal mol-1 400 μM -6.13 kcal mol-1 664 uM †Calculated at 298.15 K;
‡Calculation values from docking energy.
(R,S)-AdoMet has a good docking contrast with metallated MmuM, the best
estimated inhibition constant being 1.9 μM. The aromatic adenine ring of (R,S)-AdoMet
lies in a pronounced hydrophobic binding pocket with the exocyclic amino group
interacting with the backbone of Gly294 and Asp20 on β1. The α-amino group of (R,S)-
AdoMet is stabilized through an electrostatic interaction with Glu134 -
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carboxylic group interacts with Gln72 on β2 through a hydrogen bond (Figure 6-8AB).
Gln72 and Glu134 could be the key factors determining HMT preference for SMM or
(R,S)-AdoMet. Glu134 is located on the α’5/α’6 region connecting β3 and α3. The α’5/’6
region lies on the protein surface, and is a unique feature of HMT family proteins. This
region is disordered in our apo MmuM structure, indicating possible flexibility as a role in
protein/co-substrate interaction. Gln72 is located on the β2/α2 loop (71-79, L2) region,
which is also flexible in the MmuM apo form. L2 is critical for the substrate binding in
BHMT. rBHMT is very sensitive to cleavage between rArg86 and rLys93, but has
substantial resistance to proteolysis when bound to the Hcy mimic S-(δ-carboxybutyl)-
homocysteine332. Gln72 in MmuM is a tyrosine in rBHMT (rTyr77), and this residue
plays a role in Hcy recognition334. Gln72 in MmuM opens a larger pocket, allowing the
binding of co-substrate (R,S)-AdoMet.
Compared with (R,S)-AdoMet, the ‘undamaged’, (S,S)-AdoMet binds metallated
MmuM with a very similar estimated binding free energy and inhibition constant (Table
6-3). Most of the protein-ligand interactions remain the same; however, there is a
difference between the orientations of the S-methyl positions (Figure 6-8C). While the
S-methyl group in (R,S)-AdoMet faces the bound Hcy in a predicted, catalytically
productive mode (Figure 6-8B), in the (S,S)-AdoMet model, the activated methyl group
faces outward, pointing in the opposite direction to the thiol group of the Hcy co-
substrate. As a consequence, (S,S)-AdoMet is predicted to bind but not to serve as a
methyl donor.
Additionally, modeling of a S-ribosylMet /MmuM interaction does not result in a
binding pattern similar to that of either the AdoMet diastereomers. (S,S)-S-ribosylMet
160
docks into the substrate binding pocket in a non-productive orientation, with the α-amino
and carboxyl group near the co-substrate Hcy, and the ribosyl group on the protein
surface (Figure 6-8D). The interaction has an estimated inhibition constant of 827 μM,
which is about 500 times weaker than that for the metallated MmuM /(R,S)-AdoMet
interaction. (R,S)-S-ribosylMet binds the protein with an even higher free energy change
and a similar geometry (Figure 6-8E). As S-ribosylMet is not a productive methyl donor
for MmuM 303, we propose that the presence of the aromatic adenine ring in AdoMet
helps to correctly position the methyl donor, and thus to facilitate access of the activated
S-methyl group to the Hcy co-substrate.
Although SMM is a good methyl donor in vitro, modeling suggests that it binds
MmuM less tightly than the (R,S)-AdoMet diastereomer. In addition, our top three
docking solutions place SMM deep in a pocket, in a conformation not productive in
terms of methyl transfer (Figure 6-8F). Additional bond conformations (Rank 4, Figure 6-
8F) do show a productive solution with SMM bound in a fashion similar to that of the
Met moiety of (R,S)-AdoMet. This suggests that SMM may also access Hcy from α’5/α’6
and L2 region. As SMM is small compared to AdoMet, it is not surprising that docking
algorithms predict a relative lower binding affinity. The prediction of a higher binding
affinity for (R,S)-AdoMet than for SMM is particularly useful. Difficulties in separating
(R,S)-AdoMet and (S,S)-AdoMet along with the stability of the diastereomers, makes it
difficult to measure kinetic parameters for MmuM with (R,S)-AdoMet as methyl
donor303,320.
161
6.3.5 Specific Potassium Ion Requirement for MmuM Activity
Human hBHMT (pdb entry 4M3P) has a K+ ion near the Zn2+ site that is critical
for the catalytic activity335. hBHMT possess a much lower enzymatic activity in the K+-
deficient betaine /Hcy reaction. By comparison, our kinetic data indicates that HMT
MmuM reaction maintains the same activity with or without K+ (Table 6-2). Interestingly,
we did observe a significant, unexpected electron density located adjacent to the
substrate binding pocket in the MmuM oxidized form (Figure 6-5A). This electron
density is located near the α’2 helix and closest to NMet23 with distances of 3.3 Å, and a
contour level of 8 in the calculated mFo-DFc map. The density is also observable in
the metallated form of the protein, but has a much lower contour level. The position
does not exhibit an anomalous signal at 0.9787, ruling out the possibility of Ni2+ or Zn2+
ions. As there is a prominent dipole interaction of helix α’2 (N-terminus) pointing at the
feature, we fit the density as chloride in the deposited structure.
6.4 Conclusions and Insights
HMTs catalyze the conversion of homocysteine to methionine using S-
methylmethionine or S-adenosylmethionine as the methyl donor. HMTs play an
important role in methionine biosynthesis and are widely distributed among
microorganisms, plants, and animals. Additionally, HMTs play a role in metabolite repair
of S-adenosylmethionine by removing an inactive diastereomer from the pool. The
mmuM gene product from Escherichia coli is an archetypal HMT family protein and
contains a predicted Zn-binding motif in the enzyme active site. To summarize, we
present here the first structure of a member (MmuM) of the homocysteine
methyltransferase family, a widely distributed enzyme family that plays key roles in the
162
activated methyl cycle. Using a combination of X-ray crystallography, in vitro and in
silico assays, the work provides a structural basis for catalysis and substrate
discrimination.
6.5 Materials and Methods
6.5.1 Chemicals
Unless otherwise stated, reagents and chemicals were purchased from Fisher
Scientific, Sigma/Aldrich or Hampton.
6.5.2 Cloning, Expression, and Purification of MmuM
The gene for mmuM was amplified via polymerase chain reaction (PCR) from E.
coli K-12 MG1655 genomic DNA with the following primers: mmuM_NdeIF (GCG
CATATG TCGCAGAATAATCCGTTA), and mmuM_XhoIR (GCG CACGAG
TCAGCTTCGCGCTTTTAA). The PCR product was cleaved with the corresponding
endonucleases and then ligated into the expression vector pET30a with a C-terminal
hexa-His tag. The resultant plasmid was transformed into E. coli C43 (DE3) cells for
expression. Cultures (1 L) were grown at 37°C to an OD600=0.8, and overexpression
was initiated by adding isopropyl β-D-1-thiogalactopyranoside (final concentration 400
μM). Growth was continued for 25 h at 25°C, before the cells were harvested by
centrifugation. Cell pellets were resuspended in 25 mL of 0.5 M NaCl and 20 mM Tris-
HCl, pH 7.5, and lysed at 14,000 psi through a nitrogen-pressure microfluidizer cell (M-
110L Pneumatic). The lysate was clarified by centrifugation at 15,000g for 20 min at
4°C. MmuM was purified by immobilized metal affinity chromatography (HisPur Ni-NTA
Resin, Thermo Scientific). After binding for 1 h, the resin was washed with 4 × 10 mL of
0.5 M NaCl, 10 mM imidazole and 20 mM Tris-HCl, pH 7.5, and the bound protein was
eluted with 3 × 2 mL of 0.5 M NaCl, 250 mM imidazole and 20 mM Tris-HCl, pH 7.5.
163
The elution fraction was further purified by gel filtration chromatography (HiLoad 16/60
SuperDex-200 column, AKTA FPLC System, GE Healthcare) with buffer 150 mM NaCl,
5 mM dithiothreitol and 20 mM Tris-HCl, pH 7.5. The pooled protein was dialyzed
against 1 L 150 mM NaCl, 5mM ZnCl2, 1 mM βME and 20 mM Tris-HCl, pH 7.5 for 4 h,
then subsequently dialyzed against 1 L of 150 mM NaCl, 1 mM βME and 20 mM Tris-
HCl, pH 7.5, for an additional 4 h to remove non-specifically bound Zn2+. The protein
was centrifuged to remove precipitated protein before crystallization. Protein
concentration was determined with Bradford assay using bovine serum albumin (BSA)
as standard.
Figure 6-9. Crystallization and optimization of the MmuM. A) Crystals formed in sitting drop colored by methylene blue; B) optimization of pH and salt with glycerol as additive and, C) harvested MmuM rod-shaped single crystals.
6.5.3 MmuM Crystallization
Initial crystal screening was performed in a vapor diffusion, sitting drop format
using commercial sparse matrix screens. Small clusters of needle crystals were
identified in a condition containing 2.0 M ammonium sulfate and 0.1 M sodium acetate,
pH 4.6. Optimization of salt and pH along with microseeding were performed in hanging
drop format at 20°C. Protein (1.8 μL of a 4 mg mL-1 solution) plus 2 μL of precipitant
were mixed and balanced against 1 mL of reservoir solution. Resultant rod-shaped
164
single crystals with a size of ~20×20×100 μm were obtained in a final condition that
contained 1.6 M ammonium sulfate, 10% v/v glycerol and 0.1 M sodium acetate, pH 4.8
(Figure 6-9). Crystals of suitable size were harvested and frozen in liquid nitrogen
without additional cryoprotectant. Harvested MmuM crystals were in an oxidized metal-
free form. To obtain the apo form crystals, 10 mM dithiothreitol was added to the
reservoir solution and equilibrated with the crystal drop for 16 h. Metallated form crystals
were obtained by further soaking in 1 mM ZnCl2 and 10 mM Hcy for 5 min before
harvesting and flash freezing.
6.5.4 Data Collection and Processing, and Structure Refinement
Diffraction data were collected on beamline 21-ID-F/G of the Life Sciences
Collaborative Access Team (LS-CAT) facility at the Advanced Photon Source (APS),
Argonne National Laboratory. Data were collected at 100 K with a wavelength of 0.9786
Å, integrated, merged and scaled using XDS package156 to a resolution of 1.76 - 2.88 Å
in space group I222 or P21212, with one or two protein molecules per asymmetric unit.
The phase of MmuM in space group I222 was determined by molecular replacement
with MOLREP336 using the N-terminal structure of B12-dependent methionine
synthase323 (PDB entry 1Q7M, 26% sequence identity) from Thermotoga maritima as a
search model. The molecular replacement solution was refined by rigid-body refinement
in REFMAC5183 to a Rwork of 43% and Rfree of 51%. The refined molecular replacement
model, combined with an anomalous signal from Zn atoms, provided an interpretable
electron density map, and the atomic model was completed by several rebuilding and
refinement cycles using SHELXDE188, RESOLVE187, ARP/wARP189,
PHENIX.REFINE158 and COOT160. Zn2+ ions were added manually into the calculated
165
anomalous difference map, and occupancy and anisotropic B-factors were refined with
sequentially. Hcy was also added manually, adjacent to the Zn2+ ion. Our metallated
crystal form has extra electron density near Cys296, which we assigned as βME. A
distance restraint between SβME and SCys296 was added (2.05 Å) during the refinement.
Water molecules were placed in the structure based on manual inspection of the 2mFo-
DFc and mFo-DFc electron density maps. No TLS/NCS restraint was used during the
refinement process. The quality of the model was evaluated using a simulated
annealing composite omit map. The refined coordinates have been deposited in the
protein data bank (accession codes 5DML, 5DMN and 5DMN). Statistics on data
collection and atomic structure refinement are summarized (Table 6-1). Structural
illustrations were prepared with PyMOL161.
6.5.5 Models of Bound Methyl-Donors and Analysis
Fitting of methyl donors into the active site was conducted with AutoDock 4.2191.
The methyl donors SMM, (R,S)-AdoMet, or their mimics (S,S)-AdoMet and S-ribosylMet
were constructed and energy minimized with Spartan ’08192. The position of bound Hcy
molecule in the metallated form MmuM was held fixed as the methyl donors were fitted.
Standard algorithms and docking procedures were used for a rigid protein and a flexible
ligand in a grid covering the entire protein. The best docking poses were analyzed with
DS Visualizer 2.5 and the hydrogen bonding/π–π interactions and corresponding
estimated free energy of ligand binding (ΔG) were calculated.
6.5.6 MmuM Active Site Mutagenesis
Mutations of residues in the MmuM active site were carried out using the Q5
Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s
instructions. Oligonucleotides utilized includes: Y71F
166
(CACTGCCAGCtttCAGGCGACGC), Y71A (CACTGCCAGCgctCAGGCGACGCCGG),
Y71T (CACTGCCAGCactCAGGCGACGCCG), and Y71F/T169Y (plus
GGCCTGCGAAtacCTGCCGAATTTTTCCGAGATTG). The validity of mutagenesis was
confirmed by DNA sequencing.
6.5.7 ITC-Based Activity Assay
MmuM wild type (WT) and mutant proteins were purified as described above. WT
and mutant proteins, prepared concurrently, were each concentrated to 10.0 mg mL-1
for isothermal titration calorimetry (ITC)-based kinetics assays. Protein samples (1.0 μL,
final concentration 1.0 μM), along with 2 mM DL-Hcy, were mixed with 300 μL of freshly
prepared reaction buffer (100 mM NaCl, 100 mM KCl, 20 mM Tris HCl, pH 7.5, 1mM
DTT) and injected into the MicroCal iTC200 (Malvern) reaction cell (cell volume, 200
μL). 39.6 μL of 5.0 mM DL-SMM was dissolved in the same reaction buffer and titrated
into the cell in the single injection mode over 800 s at 30°C. Potassium-free protein
(WT) kinetics were carried in a K+ deficient media (200 mM NaCl, 20 mM Tris HCl, pH
7.5, 1mM DTT). Protein-free, blank runs were performed in a similar fashion. Heat
change during the reaction was detected and recorded. All titrations were repeated in
triplicate. The reaction kinetics parameters337 were calculated using the Origin software
package155.
6.5.8 Determination of Protein Secondary Structure
Far-UV circular dichroism spectra (CD) of purified MmuM and site-directed
mutants were recorded at 25ºC (Aviv 202 CD Spectrometer). Proteins were dialyzed
against 150 mM NaCl, 5mM ZnCl2, 1 mM βME and 20 mM Tris-HCl, pH 7.5 for 4 h after
immobilized metal affinity chromatography purification, and then dialyzed against 100
mM NaCl, 100 mM KCl, 20 mM Tris HCl, pH 7.5 and diluted to a concentration of 0.3
167
mg ml-1 for CD spectrometry. Samples were placed in 0.1 cm pathlength cuvette with
ellipticities (θ) recorded at a wavelength range of 200 - 250 nm.
168
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BIOGRAPHICAL SKETCH
Kunhua Li was born in Kunming, Yunnan province, China in 1988. He attended
Yunnan University Secondary Middle School, and Kunming No. 1 High School. Kunhua
represented Yunnan Province in the 2006 National Chemistry Olympiad Competition
and was admitted to Peking University (PKU) for the undergraduate study, where he
majored in Chemistry. Kunhua joined the key Laboratory of Polymer Chemistry under
Professor Yu-Guo Ma in 2007, and focused his undergraduate research on the
synthesis and characterization of amphiphilic supramolecules. Kunhua also worked in
the College of Pharmaceutical Science 2009-2010, which inspired his interests in
biochemistry. Kunhua received multiple awards while at PKU, including several
outstanding student awards and two grants for undergraduate research.
After obtaining Bachelor of Science (BS) degree from PKU, Kunhua moved to the
University of Florida (UF) to begin his PhD in Biochemistry, where he worked under
Professor Steven D. Bruner. While at UF, Kunhua focused on natural product pathways
and have utilized protein crystallography, organic synthesis, chemical biology, cell
biology heavily in his projects. Kunhua worked on the siderophore-based iron
acquisition and utilization project, which resulted in several publications. Kunhua
participated in several long-standing collaborative projects with Professor Andrew D.
Hanson and Professor Yousong Ding, parts of which are highlighted in this dissertation.
Kunhua also focused on the structural insight into the microviridin biosynthesis pathway
as his dissertation project. Besides graduate research, Kunhua also devoted himself in
teaching and attained Master of Science in Teaching, Chemistry in 2013. While at UF,
196
Kunhua was named outstanding international student; awarded the UF CLAS
Dissertation Fellowship; and received several travel grants.
For the further research, Kunhua desires to utilize his interdisciplinary skills to
pursue a career in using structural approaches to understand the protein-protein
interactions relate to cell signaling and regulation. Kunhua is particularly interested in
cancer related research and will join Harvard Medical School (HMS) and Dana-Farber
Cancer Institute (DFCI) as a post-doctoral fellow, where he will work with Professor
Michael J. Eck.