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PDBsum entry 2obh
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References listed in PDB file
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Key reference
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Title
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Structural, Thermodynamic, And cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group c protein.
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Authors
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J.B.Charbonnier,
E.Renaud,
S.Miron,
M.H.Le du,
Y.Blouquit,
P.Duchambon,
P.Christova,
A.Shosheva,
T.Rose,
J.F.Angulo,
C.T.Craescu.
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Ref.
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J Mol Biol, 2007,
373,
1032-1046.
[DOI no: ]
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PubMed id
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Abstract
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Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory
role in the DNA damage recognition during the first steps of the nucleotide
excision repair. This biological action is mediated by the binding to a short
fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C
(XPC) protein. This work presents a detailed structural and energetic
characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2
we obtained a high resolution (1.8 A) X-ray structure of the complex with the
peptide N847-R863 from XPC. Structural and thermodynamic analysis of the
interface revealed the existence of both electrostatic and apolar
inter-molecular interactions, but the binding energy is mainly determined by the
burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2
C-terminal domain. Binding studies with various peptide variants showed that XPC
residues W848 and L851 constitute the critical anchoring side-chains. This
enabled us to define a minimal centrin binding peptide variant of five residues,
which accounts for about 75% of the total free energy of interaction between the
two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2
binding to the integral XPC protein may be observed in living cells, and is
determined by the same interface residues identified in the X-ray structure of
the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2,
by inducing a translocation of centrin molecules from the cytoplasm to the
nucleus. The present data confirm that the in vitro structural features of the
centrin/XPC peptide complex are highly relevant to the cellular context.
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Figure 2.
Figure 2. Crystal structure of the complex
ΔN25-HsCen2/P17-XPC. (a) Global view of the two
ΔN25-HsCen2/P17-XPC complexes observed in the asymmetric unit.
The centrin molecules are represented in green and orange
ribbons. The two bound P17-XPC peptides are shown in dark blue
and red ribbons. The calcium ions present in the C-terminal
binding sites of centrin molecules are represented as magenta
(site IV) and grey (site III) balls. (b) Zoom of the
centrin/peptide interaction and position of this interface
relative to the Ca^2+ binding sites. The orientation of the
complexes in (a) and (b) is the same. (c) and (d) Electron
density maps around the Ca^2+ binding sites IV (c) and III (d).
2F[o]–F[c] maps contoured at 2σ are represented.
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Figure 3.
Figure 3. (a) The B factors of the centrin and peptide C^α
atoms in the complex ΔN25-HsCen2/P17-XPC, plotted against the
residue number. Data for the two centrin and two peptide
molecules in the crystallographic asymmetric unit (black and
red) are compared with the corresponding factors in the
structure by Thompson et al.^18 (blue and green). The helical
fragments in the protein are indicated by the shaded areas. The
empty and filled green circles indicate the free and bound Ca^2+
sites, respectively. (b) The Ramachadran plot of the complex
dimer; 95.5% and 4.5% of the residues were found in the most
favored and additional allowed regions, respectively.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
373,
1032-1046)
copyright 2007.
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Secondary reference #1
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Title
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Crystallization and preliminary X-Ray diffraction data of the complex between human centrin 2 and a peptide from the protein xpc.
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Authors
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J.B.Charbonnier,
P.Christova,
A.Shosheva,
E.Stura,
M.H.Le du,
Y.Blouquit,
P.Duchambon,
S.Miron,
C.T.Craescu.
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Ref.
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Acta Crystallogr Sect F Struct Biol Cryst Commun, 2006,
62,
649-651.
[DOI no: ]
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PubMed id
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Figure 1.
Native crystals of [Delta]N25-HsCen2 in complex with the
peptide P1-XPC obtained in 30% MPEG 500, 100 mM NaCl and 100 mM
bicine pH 9.0. The crystals were obtained by transfer of seeds
in order to limit spontaneous nucleation. Acta Crystallogr Sect
F Struct Biol Cryst Commun. 2006 July 1; 62(Pt 7): 649–651.
Published online 2006 June 10. doi: 10.1107/S1744309106019415.
Copyright [copyright] International Union of Crystallography
2006
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Figure 2.
Titration of HsCen2 with Sr^2+ ions. The isotherm, fitted to
a single-site binding model, gives stoichiometry n = 1, binding
constant K [a] = 4.0 (0.3) x 10^4 M ^[minus sign]1 and reaction
enthalpy [Delta]H = [minus sign]2720 (84) J mol^[minus sign]1.
The lower panel schematically shows the balance of the energetic
terms contributing to the binding process. Acta Crystallogr Sect
F Struct Biol Cryst Commun. 2006 July 1; 62(Pt 7): 649–651.
Published online 2006 June 10. doi: 10.1107/S1744309106019415.
Copyright [copyright] International Union of Crystallography
2006
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by the IUCr
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Secondary reference #2
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Title
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Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group c protein (xpc) from nuclear excision repair.
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Authors
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A.Yang,
S.Miron,
L.Mouawad,
P.Duchambon,
Y.Blouquit,
C.T.Craescu.
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Ref.
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Biochemistry, 2006,
45,
3653-3663.
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PubMed id
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Secondary reference #3
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Title
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The structure of the human centrin 2-Xeroderma pigmentosum group c protein complex.
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Authors
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J.R.Thompson,
Z.C.Ryan,
J.L.Salisbury,
R.Kumar.
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Ref.
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J Biol Chem, 2006,
281,
18746-18752.
[DOI no: ]
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PubMed id
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Figure 1.
FIGURE 1. Structure of human centrin-2 bound to a human
XPC-derived peptide. A, sequence of the HsCen-2 recognition site
from HsXPC structurally aligned with sequences of skeletal and
smooth muscle myosin light chain kinase (skMLCK and smMLCK) and
Kar1p from structures with calmodulin and yeast centrin (or
caltractin). The XPC peptide structure consists of residues
Asn^847-Arg^863, the HsXPC sequence underlined. Essential HsXPC
residues interacting to form  -helical coiled-coil
are indicated in red. Shaded pink are important HsXPC residues
interacting with HsCen-2. Positions numbered "1-5-8-14" of key
interfacial residues in skeletal muscle myosin light chain
kinase and smooth muscle myosin light chain kinase bound to
calmodulin are shown for comparison in purple. B, rainbow ribbon
trace of the main chains of HsCen-2 with HsXPC and two bound
Ca^2+ metals at the C-terminal domain. An ordered helical linker
separates N-terminal (blue)(Nterm) and C-terminal (red)(Cterm)
domains. The entire XPC peptide is -helix. C, two
complexes are found in the asymmetric unit. They interact solely
through bound XPC peptides that form an -helical coiled-coil
structure. D, the two independent complex structures are nearly
equivalent in overall conformation.
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Figure 3.
FIGURE 3. The two domains of HsCen-2 are compared. A
cross-eye stereo image is shown of a superposition of the
N-terminal HsCen-2 domain (blue) on the C-terminal domain (red).
The N-terminal domain exists in a closed conformation. Relative
positions of the two bound calcium atoms (dark green) bound to
EF-hands III and IV and the XPC peptide (green) with Trp^848,
Leu^851, and Leu^855 are drawn. The helices are numbered with
regard to past convention and Table 2 herein.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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