Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking.
Cys(38) and Cys(280) of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross-linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C(561)) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21. Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies.
Rausch
J W
JW
HIV Drug Resistance Program, Science Applications International Corporation, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21072, USA.
Sathyanarayana
B K
BK
Bona
M K
MK
Le Grice
S F
SF
D001386:Azides / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D017931:DNA Primers; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics
D001386:Azides; D003432:Cross-Linking Reagents; D017931:DNA Primers; D009698:Nucleoproteins; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D003545:Cysteine; C011171:4-azidophenacyl bromide