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1 Pacific Northwest National Laboratory, Molecular Biosciences Department, Richland, Washington 99352, USA
2 Pacific Northwest National Laboratory, Environmental Molecular Sciences Laboratory, Richland, Washington 99352, USA
3 Washington State University, School of Molecular Biosciences, Pullman, Washington 99164, USA
Reprint requests to: Eric J. Ackerman, Pacific Northwest National Laboratory (PNNL), Molecular Biosciences Department, P.O. Box 999, Richland, Washington 99352, USA; e-mail: eric.ackerman{at}pnl.gov; fax: (509) 376-2149.
The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length 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 recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron 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). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.
Keywords: XPA; mass spectrometry; intrinsic disorder; partial proteolysis; unstructured proteins; DNA repair
Abbreviations: ESI-FTICR, electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance • NER, nucleotide excision repair • NLS, nuclear localization signal • MBD, minimal binding domain • MW, molecular weight • PONDR, predictor of natural disordered regions • XPA, xeroderma pigmentosum group A
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