Presentazione di PowerPoint

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The Florence contribution to Inorganic
Structural Genomics
From gene to function through structure
Eukaryotes
Cox17
Gram-positive Bacteria
Gram-negative Bacteria
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Copper delivery to the trans-Golgi network in
yeast
O2-., HO-.
O2, H2O2
Cu(II), Fe(III)
cytoplasm
Cu(I)
Cu(I), Fe(II)
Cu(II)
b
a
Golgi
Ccc2
Fre1
Ftr1
ATP
Fet3
ADP
Cu(I)
Atx1
Cu(I)
Fe(III)
Fet3
Cu(I), Fe(II)
Nucleus
Ftr1
Cu(II), Fe(III)
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Cu homeostatic regulation in human
Brain
Cu
Menkes
Blood
Menkes
Blood
Liver
Bile
Wilson
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P-type ATPases
NH3
-CXXC-
Pumping machinery
-CXXC-
-CXXC-
ATP
Metal-binding domains
-CXXC-
-CXXC-
COO-
-CPC-
1
2
3
4
5
6
7
8
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Mutations in the soluble part of Menkes Ala629
to Pro (domain VI)
CYS
CYS
CYS
CYS
ALA629
PRO629
A629P
Wild-type
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The structure of bovine CCO
Mammalian CCO consists of a 13 subunit complex of
200kDa which can exist as a larger dimeric unit
Tsukihara T, Aoyama H, Yamashita E, Tomizaki T,
Yamaguchi H, Shinzawa-Itoh K, Hakashima R, Yaono
R, Yoshikawa S, Science 1995
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The CuA and CuB centers
CuA
CuB
13 Å
Cox2 contains two Cu ions in a binuclear center
Cox1 contains one Cu ion in a heme A3-Cu
binuclear site
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Proteins involved in the assembly of copper
centers of CCO
O2-., HO-.
O2, H2O2
Cu(II), Fe(III)
Cu(I), Fe(II)
Fre1
Nucleus
Cox23
Cu(I)
Cox23
Cu(I)
Cox17
CCO
Sco1
Cox17
Sco2
Cox11
Surf1
Cox19
Cox19
Mitochondria
Cu(I)
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Solution structure of Sco1 homologue from
Bacillus subtilis
?3
a1
Cys 45
?2
?6
?1
a2
?5
a3
?4
Cys 49
His 135
Cu(I)
?7
?1
?8
C
Cys 49
Cys 45
a4
His 135
N
Banci L., Bertini I., Ciofi-Baffoni S., Cantini
F., Balatri E., Structure, 2003
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Comparison with proteins having a similar fold
Apo BsSco1
TlpA, thioldisulphide oxidoreductase
HBP23, peroxiredoxin
Banci L., Bertini I., Ciofi-Baffoni S., Cantini
F., Balatri E., Structure 2003
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Genomic context of Sco1 and its paralogs
A genome-wide search of Sco1-like sequences
reveals a large number of paralog bacterial
proteins
-Neighbor genes of Sco1-like ( ) sequences
are often copper enzymes or copper-related
proteins -Domain fusions and spatial clustering
of sequences in the genomes suggest a possible
involvement of Sco1 paralogs in a variety of
biological processes other than copper
incorporation into CCO -Up to five paralog
sequences are found in a single organism the
case of Pseudomonas fluorescens
sco1
Scheme of gene associations
cytC
cox2
cox1
Cox11
Cu- oxidase
sco1
sco1
Hyp1
sco1
cytC
Cu- oxidase
sco1
sco1
CCO operon
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A copper chaperone or a thioldisulphide
oxidoreductase function ?
In vitro Sco1 binds copper(I) through the
conserved CXXXCP motif and possibly His 135,
suggesting to be involved in copper ion delivery
to the COX complex
BUT..
The structure similarity with thioredoxins
suggests that one of the functions of Sco1 may
be to reduce cysteine thiol groups in the CuA
binding site of cytochrome c oxidase.
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Assembly of the CuA center of CCO
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Assembly of the CuA center of CCO
In eukaryotes
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Solution structure of apoCox11 homologue from S.
meliloti
Cys 101
Cys 99
PDB code 1SO9
Banci L., Bertini I., Cantini F., Ciofi-Baffoni
S., Gonnelli L., Mangani S. J. Biol. Chem. 2004
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Cox11 has an immunoglobulin-like fold with a
novel type of b-strand organization
ApoCox11
A linker domain of a bacterial sialidase
A motile major sperm protein of
b- strands (gray colored) are common to all
Ig-like domains.
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Interaction with Copper(I)

• Cu(I)Cox11 is predominantly present in a dimeric
  state, ?m 14.7 ? 1.0 ns
• Cox11 binds ?1 copper(I) atom per monomer
• The EXAFS data show a sulfur coordination
  consisting of 3 S atoms at 2.27 Å and a second
  copper ion located at 2.71 Å.
• The distance of 2.7 Å is in accordance with Cu-Cu
  distances observed in doubly bridging sulfur
  complexes!

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Assembly of the CuB center of CCO
Cox17
Cu(I)Cox 11
CuB
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The reduced apoCox17
apoCox17 contains a coil-helix-coil-helix (CHCH)
domain and behaves as a molten globule
In the reduced state the helical secondary
structure is retained
Cox17 red
Cytoplasm
CCO
Sco2
Sco1
Cox11
Cu(I)
Mitochondria
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The Cu(I)Cox17
Cox17 binds 4 Cu(I) ions in a Cu4(m-S-Cys)62-
cluster and exists in a dimer/tetramer
equilibrium with a 20 ?M Kd
Cu(I)Cox17
Cytoplasm
CCO
Sco2
Sco1
Cox11
Cu(I)Cox17
Mitochondria
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The oxidized apoCox17
In the oxidized state the cysteines form two
disulfide bonds
Cox17 ox
Cytoplasm
CCO
Sco2
Sco1
Cox11
Mitochondria
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Cox17 mitochondrial import and copper binding
isomerization and copper binding
S
S
S
S
S
apoCox17 reduced
S
oxidative folding
Cu
SH
Cu1Cox17
apoCox17 oxidized
import
SH
SH
SH
SH
TOM
SH
SH
SH
SH
SH
SH
SH
copper binding
Cu4Cox17
OM
cytosol
IMS
multimerization
Arnesano, Balatri, Banci, Bertini, submitted
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Cox17 A copper chaperone for two proteins
In vitro studies show direct copper transfer from
CuCox17 to Sco1 or Cox11
Cox17
Nucleus
Cu(I)
Cox11
Cu(I)
Cu(I)
CCO
Cu(I)
Sco1
Mitochondria
Horng Y., Cobine P., Maxfield A., Carr H., Winge
D. J. Biol.Chem., 2004
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Searching Cox17 in gene-bank
At variance with Sco1, Cox17 orthologs are found
to be present only in eukaryotes !
We browsed bacterial genomes to find a protein
functionally equivalent to Cox17
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STRING Search Tool for Recurring Instances of
Neighbouring Genes
A gene neighboring search identifies a potential
Sco1 protein partner in bacteria with a
consensus motif H(M)X32HXM a good candidate to
substitute Cox 17
COG1622
Cox2 (CuA)
Cox3
COG2847
COG1845
COG1999
Sco1
COG0843
Sco1
Hyp
Cox1 (CuB)
NOG10163
Hyp2
http//www.bork.embl-heidelberg.de/STRING
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A Hyp copper protein in some bacteria
apo form
Cu(I) form
Met 86
Cu(I)
His 108
Met 75
Cu(I) form
Met 110
Banci L., Bertini I., Ciofi-Baffoni S., Kubicek,
K., Katsari, T. submitted
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Assembly of the CuA center of CCO
In prokaryotes
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Atx1Ccc2a interaction rapid metal transfer
Keq 1.5
Cu(I)-Atx1 apoCcc2a
Cu(I)-Ccc2a apoAtx1
Fast (ltms)
Fast (ltms)
Atx1-Cu(I)-Ccc2a
Copper exchange is kinetically favored via
juxtaposition of the copper donor and acceptor
metal-binding domains
Metallochaperones work like enzymes, tailoring
energetic barriers along specific reaction
pathways
Arnesano, Banci, Bertini, Cantini, Ciofi-Baffoni,
OHalloran, Huffman, J. Biol. Chem. 2001
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Proposed mechanism of copper transfer between
Atx1 and Ccc2
Cys N-term
S
SH
S
S
S
S
Cu
Cu
Cu
S
SH
S
SH
SH
S
Copper transfer occurs via a series of
intermediates where the Cu(I) ion is
three-coordinated by sulfur atoms of Cys residues
of the chaperone and the target protein
S
S
Cu
S
S
-R.A. Pufahl, C.P. Singer, K.L. Peariso, S.-J.
Lin, P. Schmidt, V.C. Culotta, J.E. Penner-Hahn
and T.V. O'Halloran, Science, 1997
-F. Arnesano, L. Banci, I. Bertini, F. Cantini,
S. Ciofi-Baffoni, D.L. Huffman and T.V.
O'Halloran, J. Biol. Chem. 2001
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Atx1Ccc2a interaction
Davg(HN)lt0.04ppm
Davg(HN)gt0.1ppm
0.04 ppmlt Davg(HN) lt0.1ppm
Not observed
Atx1
Ccc2a
Atx1
Ccc2a
90
90
Cys 15
Cys 13
Cu
Cys 16
Cys 18
Arnesano, Banci, Bertini, Cantini, Ciofi-Baffoni,
OHalloran, Huffman, J. Biol. Chem, 2001
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Docking of Atx1Ccc2a
C15
C13
C18
C16
K65
D65
K24
K28
E60
D61
K59
K62
HADDOCK Dominguez , Boelens, Bonvin, J Am Chem
Soc. 2003
Arnesano, Banci, Bertini, Bonvin, Structure, 2004
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The genomic context
In prokaryotes, comparative analysis of the
genomic context (gene fusion events,
co-occurrence of genes in operons, ) provides
functional hints In eukaryotes this is not
accomplishable with the same ease. But the
analysis of genomic context for prokaryotic
orthologs of eukaryotic proteins still yields,
through evolution, functional info Therefore,
the study of various homologs e.g. B. subitlis
CopA and CopZ, yeast Atx1 and Ccc2, A. thaliana
Atx, human Menkes protein and HAH1, .
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Solution structures of CopZ and CopAb from
Bacillus subtilis
Cu(I)-CopZ
Cu(I)-CopAb
Cys 20
Cu
Cys 17
Cu
Cys 13
Cys 16
Banci, Bertini, Del Conte et. al Biochemistry,
2001
Banci, Bertini, Ciofi-Baffoni et al. J. Mol.
Biol., 2002
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Electrostatic potential surface
Metallochaperones
Metal transporting ATPases
Atx1 (Eukaryotes)
Ccc2a (Eukaryotes)
conserved Lys/Arg close to the Cu(I) site
No charged amino acids
CopA (Bacteria)
CopZ (Bacteria)
conserved Lys/Arg close to the Cu(I) site
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(NMR) Cu(I) and apo HAH1 structures
Cu
C
C
N
N
Banci, Bertini, Ciofi, Katsari and Katsaros,
Submitted
36
Eukaryotic and bacterial ATPases
N
Bacterial organisms contains one or two soluble
domains
Eukaryotic organisms contains up to six soluble
domains
N
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Solution structure of the two N terminal domains
of apo-S46VCopA from Bacillus subtilis
Cys 17
Cys 20
Cys 88
Cys 85
In red chemical shift differences when the two
domains are separated and together
Domain a of BsCopA
Domain b of BsCopA
Banci L., Bertini I., Ciofi-Baffoni S., Gonnelli
L., Su X., J.Biol.Chem. 2003
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Interacting regions between the two domains of
apo-S46VCopA from Bacillus subtilis
Lys 121
Val 72
Gln 11
Cys 17
In green chemical shift differences when the two
domains are separated and together
Asn 119
Cys 20
Met 10
Copper binding site
Glu 122
Gln 61
Asn 104
Copper binding site
Ala 103
Cys 88
Thr 57
Cys 85
Banci L., Bertini I., Ciofi-Baffoni S., Gonnelli
L., Su X., J.Biol.Chem. 2003
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A Data Bank for SNPs

• Every 300, a base is different (SNP) for each
  individual
• This change may give rise to a different aminoacid

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Copper homeostasis-related disorders
Menkes disease (Cu deficiency) Wilson disease (Cu
toxicosis) Encephalomyopathies Familial
amyotrophic lateral sclerosis (FALS) Alzheimer
and Prion diseases
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Menkes and Wilson copper homeostasis-related
disorders
Mutations in the soluble part Menkes Ala629 to
Pro (domain VI) Wilson Gly85 to Val (domain I)
Leu492 to Ser (domain V) Gly626 to Ala (domain
VI) Total of missense/nonsense
mutations Menkes 28 Wilson 150
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Why are some mutations pathogenic? The mutated
domain, e.g. Ala629 to Pro (MNK domain VI), is
biochemically indistinguishable from the WT
The mutation may prevent the soluble
metal-binding regions from orienting correctly
with respect to the transmembrane regions to
achieve metal transfer
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Location of Sco1 homologue in Bacillus subtilis
BsSco1
plasma membrane
NH3
cytosol
trans-membrane segment
Cytochrome c oxidase
In eukaryotes Sco1 and cytochrome c oxidase
proteins are located in the mitochondrial
inter-membrane space, in Gram negative bacteria
they are anchored to the inner membrane and face
into the periplasm