Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.

Birgitta M Wöhrl,Sara H Thrall,Axel J Scheidig,Ruth Krebs,Stuart F.J Le Grice,Roger S Goody

doi:10.1074/jbc.272.28.17581

Abstract

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the beta12-beta13 hairpin (residues 227-235). It has been proposed to play a role in aligning the 3'-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66(W229A)/51 and 23-fold for mutant p66(G231A)/51, and the maximal pre-steady-state rate of nucleotide incorporation, kpol, was reduced 27-fold for p66(W229A)/51 and 70-fold for p66(G231A)/51, respectively. Mutant p66(M230A)/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Highlights

The highly conserved primer grip region in the p66 subunit of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is formed by the b12-b13 hairpin
The aim of this work was to characterize the importance of the primer grip of HIV-1 RT by kinetic analyses of four mutant RT enzymes
Residues Trp229 and Leu234 of HIV-1 p66 participate in the binding of non-nucleoside inhibitors (NNIs)

Summary

Introduction

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the b12-b13 hairpin (residues 227–235). For HIV-1 RT and for other DNA polymerases, a polymerization mechanism has been proposed where the binding of primer-template precedes the binding and incorporation of the first dNTP [5]. The first step of the reaction is thought to be the formation of a collision complex of RT and DNA that is concentration-dependent This is succeeded by a conformational change to form a tight complex, which in turn is a prerequisite for dNTP binding. A second conformational change is necessary after the initial binding of the dNTP to form a tight ternary complex This was identified as the rate-limiting step prior to nucleotide incorporation [6]. 1 The abbreviations used are: RT, reverse transcriptase; HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; p/t, primer-template hybrid; NNI, non-nucleoside inhibitor; WT, wild type § Supported by NATO Collaborative Research Grant CRG 950840. ¶ Supported by the German Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie (HIV-Verbund). i Present address: Structural Biochemistry Program, SAIC, NCI-Frederick, Cancer Research Center, Frederick, MD 21701-1201. ‡‡ Supported by National Institutes of Health Grant GM52263. 1 The abbreviations used are: RT, reverse transcriptase; HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; p/t, primer-template hybrid; NNI, non-nucleoside inhibitor; WT, wild type

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