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Crystallographic structure analysis of Chitinase enzyme from Corms of Crocus vernus

Dr. Ahmed AkremBahauddin Zakariya University, Multan

29.04.14

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Importance of Chitinase

Chitinases catalyze the hydrolysis of chitin1

Chitinases occur in a wide range of organisms, including plants, animals, viruses,

bacteria, fungi and insects, and play a variety of roles in these organisms.2

Plant chitinases are a structurally diverse group with respect to their physical

properties, enzymatic activities and localization.3

Chitin is an unbranched homopolymer of 1,4-linked N-acetyl-d-glucosamine.4

Chitin is not a component of mammalian cells; it occurs widely elsewhere in nature

and is abundant in human pathogens.

More than 75% of the industrial enzymes are hydrolases.5

1Bishop et al., 2000; 2Brunner et al., 1998; 2Hoell et al., 2005; 3Collinge et al., 1993; 4Butt & Sultan, 2010;

5Leishola et al., 2005

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1. Isolation and purification of the chitinase protein from C. vernus

2. Crystallization of the purified protein

3. X-ray diffraction data collection

3D molecular structure determination

Aims and Objectives

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Protein Crystallization

Nextal.com

Non-recombinant

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Crocus vernus

Genus Crocus belongs to family Iridaceae.

A perennial flowering plant found in Central and Southern Europe,

North Africa, Middle East, Central Asia to China.

Most expansive spice “Saffron” is from Crocus sativus L.

Chitinases & Lectins are ´´ Defense-related plant proteins``.

Plant chitinases are a structurally diverse group.3

So far no crystal structure of this plant is deposited in the Protein Data

Bank.

3Collinge et al., 1993,

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Crude protein porfile

Crocus vernus118kDa

66.2kDa

45.0kDa

35.0kDa

25.0kDa

18.4kDa

14.4kDaCrude Protein profile from corm on

SDS-PAGE

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Purified Chitinase

Final optimized Mono S chromatogram on 12% SDS-PAGE produces lectin

contaminations. Size exclusion chromatography produces 30 kDa protein bands with identical pattern

under reduced and non-reduced conditions. Partial N-terminal sequence blast showed 50% identity with the already reported

chitinases.11

T L F V E Y I G Y P L F S G V K F S D V P I N P E I T K F Q

Mono S peak Size exclusion chromatogram

L1: Reduced, L2: Non-reduced

PVDF blot

11Akrem et al., 2011

Purification techniques/Instruments

Protein characterization

• N-terminal amino acid sequencing

• MALDI/TOF Mass spectrometry

• SDS-PAGE Protein Purification

• Ammonium sulfate precipitation

• Dialysis

• Column Chromatography

• Gel filtration

• Ion exchanger columns (Cation/Anion: Isoelectric pH or pI)

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Crystallization of Chitinase

Dynamic Light Scattering (DLS) measurement of the 30 kDa purified protein showing

monodispersive and monomeric protein solution. PCT™ was performed to optimize the protein concentration. Protein crystallized at concentration of 16 mg ml -1.

Vapor diffusion method Crystal with dimensions of 0.625 × 0.370 × 0.1 mm: Scale bar, 0.5 mm. 0.1M CHES, pH 9.0 and 20% (w/v) PEG 800011

Monodisperse RH = 2.6nm Thin sheet

11Akrem et al., 2011

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Vapor Diffusion Methods

Hanging Drop Sitting Drop

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Protein Crystallization

Phase Diagram

Metastable Soluble aggregate formation but no nucleation Nucleation Critical nuclei formation and crystal growth Precipitation No nucleation. Growth of amorphous precipitate

Crystallization Machinery

Dynamic Light Scattering

(Monodispersity)

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Nanodrop (Protein quantification)

Zinsser Pipetting Robot (Digilab Genomic Solution, Germany) UV-microscope

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Protein/Salt Crystals

VIS-microscope UV-microscope 1mm size approx.

Best is to go for diffraction image

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Diffraction to 3D

Diffraction Image

3D StructureSingle CrystalX-ray Bombarment

Crystallographic Softwares

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X-ray Diffraction Data

Diffractometer Rotating anode Synchrotron: the best ultimate choice A synchrotron is a particular type of cyclic particle accelerator in which the magnetic

field (to turn the particles so they circulate) and the electric field (to accelerate the

particles) are carefully synchronized with the travelling particle beam Deutsches Elecktronen Synchrotron (DESY), Hamburg, Germany Approx. 1000 scientists from more than 30 countries around the world are working (2008) Few countries in the world are enjoying this facility

Diamond, UK ESRF, France DESY, Germany

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Crystal Mounting

Nylon loops to fish out crystals

Goniohead

X-ray gun

Cryonozzle

Microscope

Beamstop

Detectors

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Bioinformatics

Imosflm; Scala

Denzo; Scalepack

CCP4i Suite

Molecular Replacment; Molrep, Phaser, Mrbump

Homer

COOT

Refmac5

Protein Data Bank

Pdbsum

Pdb goodies

Chimera

Pymol

Auto-Rickshaw

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X-ray diffraction Data

Space group C2

Unit-cell parameters (Å,°) a = 172.3, b = 37.1, c = 126.4 Å, β = 127o

VM (Å3/ Da) 2.7

Solvent content (%) 54.2

Resolution range (Å) 25.0 - 2.1 ( 2.2 - 2.1 )

Total Reflections 14,0335 (20369)

Unique reflections 36468 (5230)

Redundancy 3.8 (3.9)

Average I/σ (I) 17.2 (6.8)

R merge* (%) 6.2 (19.4)

Data Completeness (%) 96.6 (96.0)

Table 6: Statistics for the native crystal11

Values in parentheses are for the highest resolution shell.

*R merge =∑hkl ∑i | Ii (hkl) – <I (hkl)> | ⁄ ∑hkl∑i Ii (hkl), where <I (hkl)> is the mean intensity of the observations Ii (hkl) of reflection hkl.

11Akrem et al., 2011

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mtz and pdb files

Out put of first processing is a single mtz file of few MB

Electron density map

Second important file is pdb file based on sequence homology from Protein Data Bank (PDB) like INAR for Narbonin vicia

MR strategy to solve the phase information

Sequence identity of atleast 40%

Clustalw 2, www.pdb.org

Pdbgoodies input page

Phase information and Coordinate information

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GH18 Type Chitinase

Phase problem was solved by Molecular Replacement (MR) using the narbonin structure (PDB code: 1NAR) as search model and the program Molrep.

Chitinases catalyze the hydrolysis of chitin. Chitinases occur in a wide range of organisms including plants, animals, viruses,

bacteria, fungi and insects. In glycosyl hydrolases, they are classified into family 18 and family 19 chitinases.13

Family 18, in their catalytic domain, possesses a common α, β-TIM barrel fold. Matthews’s coefficient calculations indicated two molecules per asymmetric unit.

13Henrissat & Bairoch, 1993

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TIM Barrel

Triose Phosphate Isomerase (TIM) Main feature of TIM barrel is an eight stranded parallel β-barrel making a core surrounded

by α- helices. The cavity of the TIM barrel in CVC is filled with aromatic and polar residues. The catalytic motif of CVC is directed into the cavity of TIM barrel.

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Sequence alignment between CVC, Heavime & Narbonin

16% sequence identity between CVC

and Hevamine (2HVM).14

33% sequence identity between CVC

and Narbonin. Hevamine: a plant endochitinase

isolated from rubber tree (Hevea

brasiliensis). All three proteins are sharing a TIM

barrel structure. Catalytic motif is DXDXE Two consensus sequences have been

highlighted through blue squares.

14Terwisscha van Scheltinga et al., 1996

Sequence Alignment

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Structure Alignment

The superposition of the Cα atoms of CVC with that of other members of the family

gives a rmsd of 3.5 and 3.6 Å for models 2HVM, 1NAR respectively. The catalytic motifs for all structures are on similar position in the TIM barrel. Narbonin, due to lack of an Aspartate in the catalytic motif, cannot show chitinase activity.

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Summary

A 30 kDa chitinase protein was purified from Crocus vernus corm.

Single suitable size crystals were developed from pure enzyme

Already the native Chitinase structures have been deposited at the Protein Data Bank with ID code 3SIM.

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Thanks for Thanks for kind kind

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