Structure and functional implications of the polymerase active site regionin a complex of HIV-1 RT with a double-stranded DNA template-primer and anantibody Fab fragment at 2.8 angstrom resolution

The structure of human immunodeficiency virus type 1 (HIV-1) reverse transc riptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-pr imer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has bee n refined at 2.8 Angstrom resolution. The structures of the polymerase acti ve site and neighboring regions are described in detail and a number of nov el insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located dose to the 3' terminus of the primer stran d. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymeri zation. Nearly all of the close protein-DNA interactions involve atoms of t he sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side -chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-b onding interactions with nucleotide bases of the second duplex base-pair an d is predicted to have at least one hydrogen bond with all Watson-Crick bas e-pairs at this position. Comparison of the structure of the active site re gion in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures sugge sts that template-primer binding is accompanied by significant conformation al changes of the YMDD motif that may be relevant for mechanisms of both po lymerization and inhibition by non-nucleoside inhibitors. interactions of t he "primer grip" (the beta 12-beta 13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 a nd the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. Ln the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions a nd may be involved in the transitions between different stages of the catal ytic process. The protein structural elements primarily responsible for pre cise positioning of the template-primer (including the primer grip, templat e grip, and helices alpha H and alpha I of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement o f nucleic acid and protein during polymerization. (C) 1998 Academic Press.