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. 2000 May 15;348(Pt 1):77–82.

Mutational analysis of Lys65 of HIV-1 reverse transcriptase.

N Sluis-Cremer 1, D Arion 1, N Kaushik 1, H Lim 1, M A Parniak 1
PMCID: PMC1221038  PMID: 10794716

Abstract

Amino acid Lys(65) is part of the highly flexible beta3-beta4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the epsilon-amino group of Lys(65) interacts with the gamma-phosphate of the bound deoxynucleoside triphosphate ('dNTP') substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys(65), we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys(65) in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (K(m)) for all dNTP substrates, in RT catalytic efficiency (k(cat)/K(m)) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys(65) do not affect the geometry of the loop's alpha-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys(70) and Arg(72). In particular, Glu in K65E can form a salt bridge with Arg(72), leading to the diminution of the latter residue's interaction with the alpha-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate ('ddNTP') inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.

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Selected References

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  1. Arion D., Borkow G., Gu Z., Wainberg M. A., Parniak M. A. The K65R mutation confers increased DNA polymerase processivity to HIV-1 reverse transcriptase. J Biol Chem. 1996 Aug 16;271(33):19860–19864. doi: 10.1074/jbc.271.33.19860. [DOI] [PubMed] [Google Scholar]
  2. Arts E. J., Li X., Gu Z., Kleiman L., Parniak M. A., Wainberg M. A. Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. J Biol Chem. 1994 May 20;269(20):14672–14680. [PubMed] [Google Scholar]
  3. Astatke M., Grindley N. D., Joyce C. M. Deoxynucleoside triphosphate and pyrophosphate binding sites in the catalytically competent ternary complex for the polymerase reaction catalyzed by DNA polymerase I (Klenow fragment). J Biol Chem. 1995 Jan 27;270(4):1945–1954. doi: 10.1074/jbc.270.4.1945. [DOI] [PubMed] [Google Scholar]
  4. Astatke M., Grindley N. D., Joyce C. M. How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides. J Mol Biol. 1998 Apr 24;278(1):147–165. doi: 10.1006/jmbi.1998.1672. [DOI] [PubMed] [Google Scholar]
  5. Boyer P. L., Ferris A. L., Hughes S. H. Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1. J Virol. 1992 Feb;66(2):1031–1039. doi: 10.1128/jvi.66.2.1031-1039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Clercq E. HIV resistance to reverse transcriptase inhibitors. Biochem Pharmacol. 1994 Jan 20;47(2):155–169. doi: 10.1016/0006-2952(94)90001-9. [DOI] [PubMed] [Google Scholar]
  7. Furman P. A., Fyfe J. A., St Clair M. H., Weinhold K., Rideout J. L., Freeman G. A., Lehrman S. N., Bolognesi D. P., Broder S., Mitsuya H. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8333–8337. doi: 10.1073/pnas.83.21.8333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goody R. S., Müller B., Restle T. Factors contributing to the inhibition of HIV reverse transcriptase by chain-terminating nucleotides in vitro and in vivo. FEBS Lett. 1991 Oct 7;291(1):1–5. doi: 10.1016/0014-5793(91)81089-q. [DOI] [PubMed] [Google Scholar]
  9. Gu Z., Arts E. J., Parniak M. A., Wainberg M. A. Mutated K65R recombinant reverse transcriptase of human immunodeficiency virus type 1 shows diminished chain termination in the presence of 2',3'-dideoxycytidine 5'-triphosphate and other drugs. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2760–2764. doi: 10.1073/pnas.92.7.2760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gu Z., Fletcher R. S., Arts E. J., Wainberg M. A., Parniak M. A. The K65R mutant reverse transcriptase of HIV-1 cross-resistant to 2', 3'-dideoxycytidine, 2',3'-dideoxy-3'-thiacytidine, and 2',3'-dideoxyinosine shows reduced sensitivity to specific dideoxynucleoside triphosphate inhibitors in vitro. J Biol Chem. 1994 Nov 11;269(45):28118–28122. [PubMed] [Google Scholar]
  11. Gu Z., Gao Q., Fang H., Salomon H., Parniak M. A., Goldberg E., Cameron J., Wainberg M. A. Identification of a mutation at codon 65 in the IKKK motif of reverse transcriptase that encodes human immunodeficiency virus resistance to 2',3'-dideoxycytidine and 2',3'-dideoxy-3'-thiacytidine. Antimicrob Agents Chemother. 1994 Feb;38(2):275–281. doi: 10.1128/aac.38.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harris D., Kaushik N., Pandey P. K., Yadav P. N., Pandey V. N. Functional analysis of amino acid residues constituting the dNTP binding pocket of HIV-1 reverse transcriptase. J Biol Chem. 1998 Dec 11;273(50):33624–33634. doi: 10.1074/jbc.273.50.33624. [DOI] [PubMed] [Google Scholar]
  13. Huang H., Chopra R., Verdine G. L., Harrison S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science. 1998 Nov 27;282(5394):1669–1675. doi: 10.1126/science.282.5394.1669. [DOI] [PubMed] [Google Scholar]
  14. Krebs R., Immendörfer U., Thrall S. H., Wöhrl B. M., Goody R. S. Single-step kinetics of HIV-1 reverse transcriptase mutants responsible for virus resistance to nucleoside inhibitors zidovudine and 3-TC. Biochemistry. 1997 Aug 19;36(33):10292–10300. doi: 10.1021/bi970512z. [DOI] [PubMed] [Google Scholar]
  15. Li Y., Kong Y., Korolev S., Waksman G. Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates. Protein Sci. 1998 May;7(5):1116–1123. doi: 10.1002/pro.5560070505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Li Y., Korolev S., Waksman G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 1998 Dec 15;17(24):7514–7525. doi: 10.1093/emboj/17.24.7514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Polesky A. H., Dahlberg M. E., Benkovic S. J., Grindley N. D., Joyce C. M. Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli. J Biol Chem. 1992 Apr 25;267(12):8417–8428. [PubMed] [Google Scholar]
  18. Reardon J. E. Human immunodeficiency virus reverse transcriptase. A kinetic analysis of RNA-dependent and DNA-dependent DNA polymerization. J Biol Chem. 1993 Apr 25;268(12):8743–8751. [PubMed] [Google Scholar]
  19. Reardon J. E. Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation. Biochemistry. 1992 May 12;31(18):4473–4479. doi: 10.1021/bi00133a013. [DOI] [PubMed] [Google Scholar]
  20. Sarafianos S. G., Pandey V. N., Kaushik N., Modak M. J. Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity. J Biol Chem. 1995 Aug 25;270(34):19729–19735. doi: 10.1074/jbc.270.34.19729. [DOI] [PubMed] [Google Scholar]
  21. Tantillo C., Ding J., Jacobo-Molina A., Nanni R. G., Boyer P. L., Hughes S. H., Pauwels R., Andries K., Janssen P. A., Arnold E. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. J Mol Biol. 1994 Oct 28;243(3):369–387. doi: 10.1006/jmbi.1994.1665. [DOI] [PubMed] [Google Scholar]
  22. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]

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