Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein.
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.
Charbonnier
Jean-Baptiste
JB
Laboratoire de Biologie Structurale et Radiobiologie, iBiTec-S, CEA, Commissariat à l'Energie Atomique, 91191 Gif-sur-Yvette, France.
Renaud
Emilie
E
Miron
Simona
S
Le Du
Marie Hélène
MH
Blouquit
Yves
Y
Duchambon
Patricia
P
Christova
Petya
P
Shosheva
Alexandra
A
Rose
Thierry
T
Angulo
Jaime F
JF
Craescu
Constantin T
CT
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014983:Xeroderma Pigmentosum / Q000378:metabolism
C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C086984:XPC protein, human; D002118:Calcium