title: Mutagenesis of human immunodeficiency virus reverse transcriptase p51 subunit defines residues contributing to vinylogous urea inhibition of ribonuclease H activity.
abstract: The vinylogous urea, NSC727447, was proposed to allosterically inhibit ribonuclease H (RNase H) activity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of its non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied that inhibitor binding altered active site geometry, whereas protein footprinting suggested a contribution from α-helix I residues Cys-280 and Lys-281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys-275 and Thr-286 (comprising α-helix I and portions of the neighboring αH/αI and αI/αJ connecting loops) was combined with a limited vertical scan of Cys-280. A contribution from Cys-280 was strengthened by our observation that all substitutions at this position rendered selectively mutated, reconstituted p66/p51 heterodimers ∼45-fold less sensitive to inhibition. An ∼19-fold reduced IC(50) for p51 mutant T286A coupled with a 2-8-fold increased IC(50) when intervening residues were substituted supports our original proposal of p51 α-helix I as the vinylogous urea binding site. In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product α-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. Finally, reduced DNA strand-transfer activity together with increased vinylogous urea sensitivity of p66/p51 heterodimers containing short p51 C-terminal deletions suggests an additional role for the p51 C terminus in nucleic acid binding that is compromised by inhibitor binding.
authors: HIV Drug Resistance Program, National Cancer Institute-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
authors: Suhman
authors: S
authors: Chung
authors: Jennifer T
authors: JT
authors: Miller
authors: Barry C
authors: BC
authors: Johnson
authors: Stephen H
authors: SH
authors: Hughes
authors: Stuart F J
authors: SF
authors: Le Grice
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D013876:Thiophenes / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; C539163:NSC 727447; D013876:Thiophenes; D054309:Ribonuclease H, Human Immunodeficiency Virus