Characterization of the Binding Site for Inhibitors of the HPV11 E1-E2 Protein Interaction on the E2 Transactivation Domain by Photoaffinity Labeling and Mass Spectrometry

详细信息    查看全文

• 作者：Walter Davidson ; Graham A. McGibbon ; Peter W. White ; Christiane Yoakim ; Jerry L. Hopkins ; Ingrid Guse ; David M. Hambly ; Lee Frego ; William W. Ogilvie ; Pierre Lavallé ; e ; and Jacques Archambault
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：April 1, 2004
• 年：2004
• 卷：76
• 期：7
• 页码：2095 - 2102
• 全文大小：208K
• 年卷期：v.76,no.7(April 1, 2004)
• ISSN：1520-6882

文摘

An indandione-containing class of inhibitors abrogatesDNA replication of human papillomavirus (HPV) types 6and 11 by binding reversibly to the transactivation domain(TAD) of the viral E2 protein and inhibiting its interactionwith the viral E1 helicase. To locate the binding site ofthis class of protein-protein interaction inhibitors, abenzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site ofcovalent modification of the E2-TAD was identified byproteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides byLC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminalmethyl of residue Met101. Evidence further pinpointedthe site of photoaffinity attachment to the terminal carbonatom, which is significant in providing a definitive exampleof the ability to locate photoinduced cross-linking to apolypeptide with atomic resolution using solely massspectrometric detection. The location of the inhibitorbinding site vis-à-vis the Glu39 and Glu100 residuessensitive to mutation for HPV 11 E2-TAD is discussed inrelation to the crystal structure of the E2-TAD from therelated HPV type 16.