Functional analysis of amino acid residues constituting the dNTP binding pocket of HIV-1 reverse transcriptase

In order to understand the functional implication of residues constituting the dNTP-binding pocket of human immunodeficiency virus type 1 reverse tran scriptase, we performed site-directed mutagenesis at positions 65, 72, 113, 115, 151, 183, 184, and 219, and the resulting mutant enzymes were examine d for their biochemical properties and nucleotide selectivity on RNA and DN A templates. Mutations at positions 65, 115, 183, 184, and 219 had negligib le to moderate influence on the polymerase activity, while Ala substitution at positions 72 and 151 as well as substitution with Ala or Glu at positio n 113 severely impaired the polymerase function of the enzyme. The K219A, Y 115F, and Q151M mutants had no influence on the fidelity; Y183A, Y183F, K65 A, and Q151N mutants exhibited higher fidelity on both RNA and DNA template s, while Y115A was less error-prone selectively on a DNA template. Analysis of the three-dimensional model of the enzyme-template primer-dNTP ternary complex suggests that residues Tyr-183, Lys-65, and Gln-151 may have impact on the flexibility of the dNTP-binding pocket by virtue of their multiple interactions with the dNTP, template, primer, and other neighboring residue s constituting the pocket. Recruitment of the correct versus incorrect nucl eotides may be a function of the flexibility of this pocket. A relatively r igid pocket would provide greater stringency, resulting in higher fidelity of DNA synthesis in contrast to a flexible pocket. Substitution of a residu e having multiple interactions with a residue having reduced interaction ca pability will alter the internal geometry of the pocket, thus directly infl uencing the fidelity.