lucia banci cerm and department of chemistry university of florence university of florence nmr in...
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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
15
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)