Substitution of Conserved Hydrophobic Residues in Motifs B and C of HIV-1 RT Alters the Geometry of Its Catalytic Pocket ² Bechan Sharma, Neerja Kaushik,* Kamalendra Singh, Suriender Kumar, and Virendra N. Pandey* 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, New Jersey 07103 ReceiVed June 18, 2002; ReVised Manuscript ReceiVed October 8, 2002 ABSTRACT: Recent crystallographic data suggest that a number of hydrophobic residues seen clustered between the structurally conserved RR 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 R 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 RR 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. The human immunodeficiency virus type 1 reverse tran- scriptase (HIV-1 RT) 1 is essential for the conversion of viral genomic RNA into a double-stranded DNA intermediate, which is integrated into the host cell genome (1-3). RT is a multifunctional enzyme that possesses RNA- and DNA- dependent DNA polymerase and RNase H activities (3). In the HIV-1 virion, its RT exists as a heterodimer (4), consisting of two polypeptides, i.e., p66 and its protease cleavage product p51 (4-6). The polymerase domain of p66 assumes an open conformation and folds into four distinct subdomains designated as fingers, palm, thumb, and con- nection subdomains (7). The polymerase domain of the p51 subunit is closed and acquires a different arrangement of these subdomains. This asymmetric dimeric form of HIV-1 RT is thermodynamically favorable and biologically viable in contrast to the monomeric forms of the individual subunits, which exhibit little or no catalytic activity (8). In vitro, HIV-1 RT also exists as a homodimer, exhibiting biochemical and kinetic characteristics similar to those of the heterodimeric enzyme (9). Although the p51 subunit has also been suggested to exist as a homodimer, we have recently demonstrated that it mainly exists in a monomeric form (10). The availability of high-resolution X-ray crystal structures of HIV-1 RT in unliganded form (11, 12) and liganded with non-nucleoside inhibitors (7, 13-16), RNA-DNA hybrid (17), double-stranded DNA (18, 19), and with DNA-dNTP (20) has greatly facilitated the analysis and understanding of the various mechanistic aspects of this enzyme. These structural studies in conjunction with biochemical evalua- tion of recombinant HIV-1 RT variants have led to the identification of mutations impacting substrate dNTP bind- ing and/or selection, drug resistance, and fidelity para- meters. For instance, the interaction of DNA polymerases ² This research was supported by a grant from the National Cancer Institute, National Institutes of Health (CA72821). * To whom correspondence should be addressed. Telephone: (973) 972-0660 and (973) 972-8653. Fax: (973) 972-8657 and (973) 972- 5594. E-mail: pandey@umdnj.edu and kaushik@umdnj.edu. 1 Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis; DTT, dithiothreitol; PMSF, phenylmethane- sulfonyl fluoride; IPTG, isopropyl -thiogalactopyranoside; poly(rA)‚ (dT) 18, polyriboadenylic acid annealed with (oligodeoxythymidylic acid)18; dNTP, deoxyribonucleoside triphosphate; dATP, dCTP, dGTP, and dTTP, nucleoside triphosphates of deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine, respectively; HIV-1 RT, human immunodeficiency virus type 1 reverse transcriptase; IMAC, im- mobilized metal affinity chromatography; U5-PBS HIV-1 RNA tem- plate, HIV-1 genomic RNA template corresponding to the primer binding sequence region; U5-PBS HIV-1 DNA template, DNA template corresponding to the U5-PBS HIV-1 genomic RNA sequence; WT, wild type. A, D, E, F, I, L, M, R, T, V, W, and Y represent single- letter codes for Ala, Asp, Glu, Phe, Ile, Leu, Met, Arg, Thr, Val, Trp, and Tyr, respectively. 15685 Biochemistry 2002, 41, 15685-15697 10.1021/bi026311z CCC: $22.00 © 2002 American Chemical Society Published on Web 12/04/2002