The carboxy-terminal domain of xeroderma pigmentosum complementation group C protein, involved in TFIIH and centrin binding, is highly disordered.
Xeroderma pigmentousum group C protein (XPC) is involved in the first step of nucleotide excision repair, with multiple functional roles including DNA damage recognition and recruitment of the repair machinery. This human protein of 940 residues forms a strong heterotrimeric complex with Rad23B and centrin 2. The structure of XPC is actually not known, and lack of significant sequence homology with proteins from structural data bases precludes any relevant prediction. Here, we present the molecular and structural characterization of a C-terminal fragment of XPC (C-XPC: 126 residues, 815-940), which was shown to be involved in centrin 2 and TFIIH binding. C-XPC may be highly expressed in E. coli, but because of its limited solubility it was purified under 6 M urea. Using bioinformatics tools, and a combination of several experimental methods (circular dichroism, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering), we show that C-XPC has a highly flexible structure under native physiological conditions, with a propensity to form helical secondary structures. Isothermal titration calorimetry experiments show that the C-XPC fragment binds human centrin 2 with high affinity and a 1:1 stoichiometry. NMR analysis indicates that the physical interaction between C-XPC and centrin 2 induces only minor conformational changes into XPC, localized around the 17-mer segment (847-863), showed to be critically involved in the centrin binding.
Miron
Simona
S
Integrative Imaging Unit, INSERM U759/Institut Curie Centre de Recherche, Centre Universitaire Paris-Sud, Bâtiment 112, 91405 Orsay, France. Simona.Miron@curie.u-psud.fr
Duchambon
Patricia
P
Blouquit
Yves
Y
Durand
Dominique
D
Craescu
Constantin T
CT
D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002151:Calorimetry; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D053838:Scattering, Small Angle; D013056:Spectrophotometry, Ultraviolet; D051758:Transcription Factor TFIIH / Q000737:chemistry*; D014961:X-Ray Diffraction
C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051758:Transcription Factor TFIIH; C086984:XPC protein, human