REFINED STRUCTURE OF HIV-1 REVERSE TRANSCRIPTASE COMPLEXED WITH A DOUBLED-STRANDED DNA AND AN ANTIBODY FAB FRAGMENT AT 2.8 Å RESOLUTION. Jianping Ding¹, Kalyan Das¹, Yu Hsiou¹, Alfredo Jacobo-Molina¹, Stephen H. Hughes², and Edward Arnold¹. ¹CABM & Rutgers University, Piscataway, NJ 08854; ²ABL-Basic Research Program, NCI, Frederick, MD 21702

The structure of a ternary complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a double-stranded DNA template-primer (dsDNA), and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 Å resolution. The detailed structures of the bound DNA, the RT polymerase and RNase H active sites, and the protein-nucleic acid interactions will be described. The first 5 duplex base pairs of the DNA near the polymerase active site conform to A-form geometry; an approximately 40deg. bend occurs smoothly over the next 4 base pairs; and the following 9 base pairs conform to B-form geometry. The bend at the A-form/B-form junction occurs in the vicinity of the [[alpha]]H and [[alpha]]I helices of the p66 thumb, which are partly inserted into a widened minor groove. The polymerase active site is composed of structural elements from both protein and nucleic acid which can influence the recognition and incorporation of incoming dNTPs. Asp110, Asp185, and Asp186 are highly conserved residues at the polymerase active site that probably function in both dNTP binding and catalysis. Tyr183 and Met184, which are part of the YMDD motif characteristic of retroviral RTs, interact with the primer 3'-terminal nucleotide. Residues that do not directly interact with the template-primer but are expected to form part of the dNTP-binding site include Asp113, Tyr115, Phe116, Gln151, Phe160, and Lys219. The [[beta]]12-[[beta]]13 hairpin (also known as the "primer grip") interacts with the sugar-phosphate backbone of the primer terminus via main chain atoms of Met230 and Gly231, which are also strongly conserved in all RNA-dependent polymerases. Amino acid residues forming part of the "template grip" (that makes close contacts with the template strand) include Asp76, Glu89, Gly93, Ile94, Gln151, Gly152, Trp153, Lys154, and Pro157. The RNase H active site, defined by residues Asp447, Glu478, Asp498, and Asp549, has a structure similar to those found in crystal structures of the isolated RNase H domain. The implications of these structural findings for enzymatic reactions and structure-based drug design are discussed in light of biochemical and genetic data.