Identification of intrinsic order and disorder in the DNA repair protein XPA

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution struct ures of a human XPA fragment comprising approximately 40% of the full-lengt h protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full -length XPA, we performed time-resolved trypsin proteolysis on active recom binant Xenopus XPA (xXPA), The resulting proteolytic fragments were analyze d by electrospray ionization interface coupled to a Fourier transform ion c yclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). More over, the mass spectrometric data allowed the assignment of multiple xXPA f ragments not resolvable by SDS-PAGE, The neural network program Predictor o f Natural Disordered Regions (PONDR) applied to xXPA predicted extended dis ordered N- and C-terminal regions with an ordered internal core. This predi ction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intr insically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q8 5-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structu re for the corresponding XPA fragment.