title: Substitution of conserved hydrophobic residues in motifs B and C of HIV-1 RT alters the geometry of its catalytic pocket.
abstract: Recent crystallographic data suggest that a number of hydrophobic residues seen clustered between the structurally conserved alphabetabetaalpha motif of the palm subdomain and at the junction of palm and fingers subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) provide an optimal geometry to the alphabeta sandwich of the palm subdomain, which harbors the catalytic site and the primer-binding grip region. This region has also been implicated in binding to the non-nucleoside RT inhibitors. We have evaluated the impact of conserved and nonconserved amino acid substitutions at four hydrophobic positions in this region of HIV-1 RT, in the context of their biochemical characteristics. The residues that have been analyzed include Ile-167, Leu-187, and Val-189 which are located within the alphabetabetaalpha motif, while Trp-153 lies next to the conserved LPQG motif, at the juncture of the palm and fingers subdomains. Our results show that all substitutions at I167 with the exception of I167T were deleterious to enzyme function in contrast to substitutions at V189 which enhanced the enzymatic activity. Ala substitution at residues W153 and L187 also substantially hindered the polymerase function of the enzyme. Further analysis revealed that the defective mutant derivatives of I167 were substantially impaired in their apparent dNTP binding abilities, thereby impacting the geometry of the dNTP binding pocket. The extent of misinsertion and misincorporation was higher in the case of RT variants of W153 and V189, specifically on a DNA template. Interestingly, none of the mutant derivatives of these residues were resistant to nucleoside inhibitors. A salient finding was that all nonconserved mutants of these residues exhibited hypersensitivity to nevirapine. We have analyzed these findings and their significance in the context of the HIV-1 RT structure and propose that these residues exert their effect via their indirect interactions with the template-primer through residues in their vicinity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for the Study of Emerging and Re-Emerging Pathogens, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA.
authors.forename: Bechan
authors.initials: B
authors.lastname: Sharma
authors.forename: Neerja
authors.initials: N
authors.lastname: Kaushik
authors.forename: Kamalendra
authors.initials: K
authors.lastname: Singh
authors.forename: Suriender
authors.initials: S
authors.lastname: Kumar
authors.forename: Virendra N
authors.initials: VN
authors.lastname: Pandey
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D003853:Deoxyribonucleosides / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry; D015224:Dideoxynucleosides / Q000737:chemistry; D005822:Genetic Vectors / Q000138:chemical synthesis; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019829:Nevirapine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000302:isolation & purification; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003853:Deoxyribonucleosides; D003854:Deoxyribonucleotides; D015224:Dideoxynucleosides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; D054303:HIV Reverse Transcriptase