Lucia BanciLucia BanciCERM CERM and Department of Chemistryand Department of Chemistry

University of FlorenceUniversity of Florence

NMR in SPINENMR in SPINEStructural proteomics of Structural proteomics of

metalloproteinsmetalloproteins

Structural Genomics Structural Genomics From the sequence…to the function From the sequence…to the function

through structurethrough structure

Sequence Function

Gene knock out + protein localizationGene knock out + protein localization + bioinformatic tools + biochemical assays+ bioinformatic tools + biochemical assays

Structure

Structural genomics: Structural genomics: from gene to structure of the proteins,from gene to structure of the proteins, a complete coverage of the genomesa complete coverage of the genomes

~~ 2300 Human zinc-binding proteins

Zinc

116 Cu-binding proteins structures from any organism

in PDB

They provide 97 distinct patterns

~~ 850 Human copper-binding proteins

Copper

Searching for metalloproteins in genebanksSearching for metalloproteins in genebanks

610 Zn-binding proteins structures from any organism

in PDB

They provide 490 distinct patterns

A bioinformatic analysis based on Metal Binding Patterns A bioinformatic analysis based on Metal Binding Patterns (MBP) and sequence identity around the MBP(MBP) and sequence identity around the MBP

foundfound

0

5

10

15

20

25

30

35

40

structuressolved

1993-4 1995-6 1997-8 1999-0 2001-2 2003-4MetalloproteinMetalloprotein structures solved:structures solved:ca. 15 iron-sulfur proteinsca. 30 heme-proteinsca. 50 copper + zinc + calcium proteinsca. 50 copper + zinc + calcium proteins

Structural biology by NMR in FlorenceStructural biology by NMR in Florence

Start of SPINE

Copper in Cytochrome c Oxidase assembly

Today I talk of… Today I talk of…

Fre1

Cu(I)

Cu(II), Fe(III)

Ctr1

Cu(I), Fe(II)

O2, H2O2

O2-., HO-.

Proteins involved in the assembly of copper centers of CCOProteins involved in the assembly of copper centers of CCO

Cu(I)

MitochondriaMitochondria

CCOCCOSco2Sco2

Cox17Cox17

Cox11Cox11

Cu(I)

Surf1Surf1

Cox17Cox17

Cox19Cox19 Cox19Cox19

Sco1Sco1

Cox23Cox23Cox23Cox23

Sco1: Sco1: in vivoin vivo data data

1. Sco1 cells are respiratory-deficient. Excess Cu(II), or overexpression of either Cox17 or Sco2, cannot correct the Sco1-associated deficiency

2. In the absence of Sco1, Cox2 subunit is unstable and degraded

3. Deletion of Sco in B. subtilis depresses expression of CcO but not menaquinol oxidase (no CuA site)

4. Sco1 contains a potential metal-binding motif CXXXC, and both cysteines are essential for function

5. Eukaryotic organisms have two homologous proteins, Sco1 ad Sco2, both dimeric, while they are monomeric in prokaryotes

Solution structure of Solution structure of B. subtilisB. subtilis Sco1 homologue Sco1 homologue

N

C

1

23

4

1 1

5

6

7

8

Cys 45

His 135

Cys 49

Banci L., Bertini I., Ciofi-Baffoni S., Cantini F., Balatri E., Structure, 2003

Cys 49

Cys 45

His 135

+ Cu(I)

Sco1 also binds type2 Cu(II) (also EPR and EXAFS data)

Apo BsSco1

TlpA, thiol:disulphide oxidoreductase

HBP23, peroxiredoxin

Comparison with proteins having a similar foldComparison with proteins having a similar fold

Banci L., Bertini I., Ciofi-Baffoni S., Cantini F., Balatri E., Structure 2003

Which is the functional role of Sco1?Which is the functional role of Sco1?

Copper binding ability suggests that Sco1 is

copper chaperone

Structural data suggest that Sco1 can have a

disulfide reductase activity

(thioredoxin fold) on the protein partner CuA site

maybe both??

Role of Cox17 as a Mitochondrial Copper Role of Cox17 as a Mitochondrial Copper ChaperoneChaperone

Horng, Cobine, Maxfield, Carr, Winge JBC 2004

Sco1 inserts two copper ions to CuA site

Cox11 inserts one copper ion to CuB site

in vitro and cytosolic data confirm Cu transfer to Sco1 and Cox11 Yeast cells lacking the COX17 gene are respiratory deficient. Cell respiration is recovered by copper addition

Cox17, no DTT

Cox17, 1 mM DTT

Cox17 ox: 2 S-S bonds and 2 SHon the basis of 13C carbon shifts

Cox17, 10 mM DTT

Cox17 red: 6 SH on the basis of 13C carbon shifts

Mixture of oxidized and reduced protein

TheThe folding properties of apoCox17folding properties of apoCox17

Proton-less Proton-less 1313C direct detection spectra are essential for C direct detection spectra are essential for assignment of partially unfolded proteinsassignment of partially unfolded proteins

13C-13C CBCACO

13C

13CO

The reduced apoCox17The reduced apoCox17

apoCox17 contains a coil-helix-coil-helix (CHCH) domain and behaves as a molten globule

MitochondriaMitochondria

CCOCCOSco1Sco1

Sco2Sco2Cox11Cox11

Cu(I)

In the reduced state the helical secondary structure is retained

CytoplasmCytoplasmCox17 red

Beers, Glerum, Tzagaloff, J Biol Chem 1997

Arnesano,Balatri,Banci, Bertini, Structure 2005

The oxidized apoCox17The oxidized apoCox17

MitochondriaMitochondria

CCOCCOSco1Sco1

Sco2Sco2Cox11Cox11

In the oxidized state the cysteines form two disulfide bonds It can bind 1 eq of Cu(I)

Cox17 ox

Arnesano, Balatri, Banci, Bertini, Structure ,2005

CytoplasmCytoplasm

X

The disulfide isomerization of ox Cox17 The disulfide isomerization of ox Cox17 upon Cu(I) bindingupon Cu(I) binding

Arnesano, Balatri, Banci, Bertini, Winge, Structure 2005

CX9C

The Cu(I)The Cu(I)44Cox17Cox17

MitochondriaMitochondria

CCOCCOSco1Sco1

Sco2Sco2Cox11Cox11

CytoplasmCytoplasm

Cu(I)4Cox17

Reduced Cox17 binds 4 Cu(I) ions in a Cu4(-S-Cys)62- cluster and exists in a

dimer/tetramer equilibrium with a 20 M Kd

Cu(I)Cox17Cu(I)Cox17

Palumaa,Kangur,Voronova,Sillard, Biochem. J. 2004

Arnesano,Balatri,Banci, Bertini, Structure 2005

SH SHSH

SHapoCox17 reduced

importTOM

SH SHSH

SH

cytosolOM

IMS IMmatrix

apoCox17 oxidized

SH SH

SH SH

CuCu

CuCu

SS

SS

SS

oxidative folding

copper binding

Cu4Cox17

CuCu

CuCu

SS

SS

SS

CuCu

CuCu

SS

SS

SS

CuCu

CuCu

SS

SS

SS

CuCu

CuCu

SS

SS

SS

multimerization

S

S

S

S

SHSH

S

S

S

S

SS

isomerization andcopper binding

CuCu1Cox17

Cox17 mitochondrial import and copper bindingCox17 mitochondrial import and copper binding

Arnesano,Balatri,Banci, Bertini, Structure 2005

Searching Cox17 in gene-bankSearching Cox17 in gene-bank

At variance with Sco1, Cox17 orthologs are found only in eukaryotes !

We browsed bacterial genomes to find a protein functionally equivalent to Cox17

Gene neighborhood analysis of cytochrome c oxidase accessory proteins

A gene neighboring search identifies (Hyp1) a potential Sco1 protein partner in bacteria with a consensus motif H(M)X32HXM: a good candidate to substitute Cox17

PA

VV

SO,Mdeg, AvinPP

RS

Pflu

AqPsyr

PSPTO

Reut

XCC

Bcep

Magn

Cj, VCA

VPA

CC

XAC

NMA, NMB

DR DR1886 DR1885

Cox2

Cox1

Cox11

Hyp1

Sco1

CuA CuB

Sco1/Hyp1: - Co-occurrence

- Conserved neighborhood

Cyt c

Arnesano, Banci, Bertini, Martinelli, J. Proteom Research, 2005

A Sco1-related copper proteinA Sco1-related copper protein

Banci L., Bertini I., Ciofi-Baffoni S., Katsari E., Kubicek K., PNAS 2005

The charge-state distribution of the ESI-MS peaks and CD spectra indicates that copper(I) binding produces a more compact conformational state than the apo form

Met 75

His 108Met 110

apo form

Met 86

Cu(I) form

A gene neighboring search identifies a potential Sco1 protein partner in bacteria with a consensus motif H(M)X32HXM: it may substitute Cox 17?

Cu(I)

His 108

Met 86

Met 110

Cu(I)

4

5’

Met 75

N

C

1

2

3

44’

5 5’

6

7

M 41

3

2

C

N

4’

55’

6

7

HxM

M

DR1885

6

5

2

4’

1

71’

34C

N

N

C

3

4

5

6

7

2

1

1’

4’

MxxMxHxxMCopC

Cu(I) site Cu(II) site

cupredoxin fold

DR1885 adopts a fold reminiscent of other

bacterial extra-cytoplasmic copper

proteins

DR1885 might be an extra-cytoplasmic

chaperone specific for copper(I)

Banci, Bertini, Ciofi-Baffoni, Kubicek, et. al. PNAS, 2005

Comparison with proteins with similar foldComparison with proteins with similar fold

Arnesano, Banci, Bertini, Mangani, Thompsett, PNAS, 2003

Assembly of the CuAssembly of the CuAA center of CCO center of CCO

Sco2

CuA

Sco1 Cu(I)

In prokaryotsSco1-partner

In eukaryotesCox17

Cu(I)

ApoCox11

A linker domain of a bacterial sialidase

A motile major sperm protein of

Ascaris suum strands (gray colored) are common to all Ig-like domains.

Cox11 has an immunoglobulin-like fold with a novel type Cox11 has an immunoglobulin-like fold with a novel type of of -strand organization-strand organization

Cu(I) binding in Cox11Cu(I) binding in Cox11

Cys 99

Cys 101

Cu(I)Cox11

Copper binding induces protein dimerizationCopper binding induces protein dimerizationThe importance of EXAFS in NMR structure determination of metalloproteins The importance of EXAFS in NMR structure determination of metalloproteins

8970 8980 8990 9000 9010 9020 90300.0

0.2

0.4

0.6

0.8

1.0

1.2

No

rmal

ized

Flo

ure

scen

ce

Energy (eV)

2 4 6 8 10 12 14 16

-6

-4

-2

0

2

4

6

(k)

*k3

k (Å-1)

0 1 2 3 4 5 60

5

10

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20

25

30

35

FT

mag

nit

ud

e

r (Å)

3S atoms at 2.27 Å and a

second copper ion located at

2.71 Å

Banci L., Bertini I., Cantini F., Ciofi-Baffoni S., Gonnelli L., Mangani S. J. Biol. Chem. 2004

Cu(I)Cox 11

Assembly of the CuAssembly of the CuBB center of CCO center of CCO

Cox17

CuB

Cu(I)

Cu(I)

From gene to function through structureFrom gene to function through structureGram-negative Bacteria

The Florence contribution within SPINE projectThe Florence contribution within SPINE project

Gram-positive Bacteria

Cox17

SOD-like

CadA

Toxic Cadmium

ADP

ATP

From gene to function through structureFrom gene to function through structure The Florence contribution within SPINE projectThe Florence contribution within SPINE project

Cox17

Cu8MT

-Parvalbumin

-Parvalbumin

MMP12

apoS100A13

CaS100A13

Mitoch

Calmodulin

Cox17Sco1

I THANK YOU FOR YOUR INTEREST AND ATTENTION!

THE ENDTHE END

PDB code: 1SO9

Banci L., Bertini I., Cantini F., Ciofi-Baffoni S., Gonnelli L., Mangani S. J. Biol. Chem. 2004

Cys 99 Cys 101

Solution structure of Cox11 homologueSolution structure of Cox11 homologue

from from S. melilotiS. meliloti

Cys 99 Cys 101Cu(I)