A Positively Charged Side Chain at Position 154 on the Beta8-alphaE Loop of HIV-1 RT is Required for Stable Ternary Complex Formation

Lys154 is the only positively charged residue located in the VLPQGWK motif on the beta8-alphaE loop at the junction of the fingers and palm subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Some of the conserved residues in this motif are critical for RT function, while others have been shown to confer nucleoside drug resistance and fidelity to the enzyme. In order to understand the functional implication of this positively charged residue, we carried out site-directed mutagenesis at position 154 and biochemically characterized the mutant enzymes. Mutants carrying negatively charged side chains (K154D and K154E) were severely impaired in their polymerase function, while those with hydrophobic side chains (K154A and K154I) were moderately affected. Analysis of the binary complexes formed by these mutants revealed that all the mutant derivatives retained their ability to form an enzyme template primer (E-TP) binary complex similar to the wild-type enzyme. In contrast, their ability to form stable E-TP-dNTP ternary complexes varied greatly and was dependent on the nature of the side chain at position 154. The conservative Lys-->Arg mutant was not affected in its ability to form a stable ternary complex, while those carrying non-polar or negatively charged side chains were significantly impaired. The apparent K(d [dNTP]) values for these non-conservative mutants were approximately 16- to 400-fold higher than the wild-type enzyme, indicating that a positively charged side chain at position 154 may be required for efficient formation of a stable ternary complex. Interestingly, all the mutant derivatives of Lys154 were completely resistant to a nucleoside analog inhibitor, 3'-dideoxy 3'-thiacytidine (3TC), implying that Lys154 may play a role in conferring 3TC sensitivity to HIV-1 RT. These findings are discussed in the context of the binary and ternary complex crystal structures of HIV-1 RT.