Biochemical and structural domain analysis of xeroderma pigmentosum complementation group C protein.
XPC is a 940-residue multidomain protein critical for the sensing of aberrant DNA and initiation of global genome nucleotide excision repair. The C-terminal portion of XPC (residues 492-940; XPC-C) has critical interactions with DNA, RAD23B, CETN2, and TFIIH, whereas functional roles have not yet been assigned to the N-terminal portion (residues 1-491; XPC-N). In order to analyze the molecular basis for XPC function and mutational defects associated with xeroderma pigmentosum (XP) disease, a series of stable bacterially expressed N- and C-terminal fragments were designed on the basis of sequence analysis and produced for biochemical characterization. Limited proteolysis experiments combined with mass spectrometry revealed that the full XPC-C is stable but XPC-N is not. However, a previously unrecognized folded helical structural domain was found within XPC-N, XPC(156-325). Pull-down and protease protection assays demonstrated that XPC(156-325) physically interacts with the DNA repair factor XPA, establishing the first functional role for XPC-N. XPC-C exhibits binding characteristics of the full-length protein, including stimulation of DNA binding by physical interaction with RAD23B and CETN2. Analysis of an XPC missense mutation (Trp690Ser) found in certain patients with XP disease revealed that this mutation is associated with a diminished ability to bind DNA. Evidence of contributions to protein interactions from regions in both XPC-N and XPC-C along with recently recognized homologies to yeast PNGase prompted construction of a structural model of a folded XPC core. This model offers key insights into how domains from the two portions of the protein may cooperate in generating specific XPC functions.
Bunick
Christopher G
CG
Department of Biochemistry, Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA.
Miller
Michael R
MR
Fuller
Brian E
BE
Fanning
Ellen
E
Chazin
Walter J
WJ
D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D014157:Transcription Factors; C086984:XPC protein, human; D004247:DNA