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.
An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.
Davidson
Walter
W
Research and Development, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Québec, Canada H7S 2G5.
McGibbon
Graham A
GA
White
Peter W
PW
Yoakim
Christiane
C
Hopkins
Jerry L
JL
Guse
Ingrid
I
Hambly
David M
DM
Frego
Lee
L
Ogilvie
William W
WW
Lavallée
Pierre
P
Archambault
Jacques
J
D000998:Antiviral Agents / Q000737:chemistry* / Q000378:metabolism; D001577:Benzophenones / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D027383:Papillomaviridae / Q000737:chemistry*; D019920:Photoaffinity Labels / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D000998:Antiviral Agents; D001577:Benzophenones; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; C117355:E2 protein, Human papillomavirus type 11; D004791:Enzyme Inhibitors; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D014764:Viral Proteins; D014357:Trypsin