HIV-1 Reverse Transcriptase (RT) is a multifunctional enzyme responsible for the transcription of the RNA genome of the HIV virus into DNA suitable for incorporation within the DNA of human host cells. Its crucial role in the viral life cycle has made it one of the major targets for antiretroviral drug therapy. The Non-Nucleoside RT Inhibitor (NNRTI) class of drugs binds allosterically to the enzyme, affecting many aspects of its activity. We use both coarse grained network models and atomistic molecular dynamics to explore the changes in protein dynamics induced by NNRTI binding. We identify changes in the flexibility and conformation of residue Glu396 in the RNaseH primer grip which could provide an explanation for the acceleration in RNaseH cleavage rate observed experimentally in NNRTI bound HIV-1 RT. We further suggest a plausible path for conformational and dynamic changes to be communicated from the vicinity of the NNRTI binding pocket to the RNaseH at the other end of the enzyme.
References
[1]
UNAIDS (Joint United Nations programme on HIV/AIDS). Available online: http://www.unaids.org (accessed on 1 May 2012).
[2]
Alfano, M.; Poli, G. The HIV life cycle: Multiple targets for antiretroviral agents. Drug Des. Rev. Online 2004, 1, 83–92, doi:10.2174/1567269043480717.
[3]
The Research Collaboratory for Structural Bioinformatics (RCSB). Protein Data Bank. Available online: http://www.rcsb.org/pdb (accessed on 1 May 2012).
[4]
Sarafianos, S.G.; Clark, A.D.; Das, K.; Tuske, S.; Birktoft, J.J.; Ilankumaran, P.; Ramesha, A.R.; Sayer, J.M.; Jerina, D.M.; Boyer, P.L.; et al. Structures of HIV-1 reverse transcriptase with pre-and post-translocation AZTMP-terminated DNA. EMBO J. 2002, 21, 6614–6624.
[5]
Tuske, S.; Sarafianos, S.G.; Clark, A.D.; Ding, J.; Naeger, L.K.; White, K.L.; Miller, M.D.; Gibbs, C.S.; Boyer, P.L.; Clark, P.; et al. Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir. Nat. Struct. Mol. Biol. 2004, 11, 469–474.
[6]
Hsiou, Y.; Ding, J.; Das, K.; Clark, A.D.; Boyer, P.L.; Lewi, P.; Janssen, P.A.; Kleim, J.P.; R？sner, M.; Hughes, S.H.; et al. The Lys103Asn mutation of HIV-1 RT: A novel mechanism of drug resistance. J. Mol. Biol. 2001, 309, 437–445, doi:10.1006/jmbi.2001.4648.
[7]
Lindberg, J.; Sigurdsson, S.; L¨owgren, S.; Andersson, H.O.; Sahlberg, C.; Nor′een, R.; Fridborg, K.; Zhang, H.; Unge, T. Structural basis for the inhibitory efficacy of efavirenz (DMP-266), MSC194 and PNU142721 towards the HIV-1 RT K103N mutant. Eur. J. Biochem. 2002, 269, 1670–1677.
[8]
Wang, J.; Smerdon, S.J.; J¨ager, J.; Kohlstaedt, L.A.; Rice, P.A.; Friedman, J.M.; Steitz, T.A. Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 7242–7246.
[9]
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, 282, 1669–1675.
[10]
Jacobo-Molina, A.; Ding, J.; Nanni, R.G.; Clark, A.D.; Lu, X.; Tantillo, C.; Williams, R.L.; Kamer, G.; Ferris, A.L.; Clark, P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 ？resolution shows bent DNA. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 6320–6324.
[11]
Peletskaya, E.N.; Kogon, A.A.; Tuske, S.; Arnold, E.; Hughes, S.H. Nonnucleoside inhibitor binding affects the interactions of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase with DNA. J. Virol. 2004, 78, 3387–3397, doi:10.1128/JVI.78.7.3387-3397.2004.
[12]
Sarafianos, S.G.; Das, K.; Tantillo, C.; Clark, A.D.; Ding, J.; Whitcomb, J.M.; Boyer, P.L.; Hughes, S.H.; Arnold, E. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA. EMBO J. 2001, 20, 1449–1461.
[13]
Hsiou, Y.; Ding, J.; Das, K.; Clark, A.D.; Hughes, S.H.; Arnold, E. Structure of unliganded HIV-1 reverse transcriptase at 2.7 ？ resolution: Implications of conformational changes for polymerization and inhibition mechanisms. Structure 1996, 4, 853–860, doi:10.1016/S0969-2126(96)00091-3.
[14]
Jacobo-Molina, A.; Arnold, E. HIV reverse transcriptase structure-function relationships. Biochemistry 1991, 30, 6351–6356, doi:10.1021/bi00240a001.
[15]
Baltimore, D. Viral RNA-dependent DNA polymerase. 1970. Biotechnology 1992, 24, 3–5.
[16]
Esnouf, R.; Ren, J.; Ross, C.; Jones, Y.; Stammers, D.; Stuart, D. Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. Nat. Struct. Biol. 1995, 2, 303–308, doi:10.1038/nsb0495-303.
[17]
Kohlstaedt, L.A.; Wang, J.; Friedman, J.M.; Rice, P.A.; Steitz, T.A. Crystal structure at 3.5 ？ resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 1992, 256, 1783–1790.
[18]
Sluis-Cremer, N.; Temiz, N.A.; Bahar, I. Conformational changes in HIV-1 reverse transcriptase induced by nonnucleoside reverse transcriptase inhibitor binding. Curr. HIV Res. 2004, 2, 323–332, doi:10.2174/1570162043351093.
[19]
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, 243, 369–387, doi:10.1006/jmbi.1994.1665.
[20]
Dash, C.; Scarth, B.J.; Badorrek, C.; Gotte, M.; Le Grice, S.F.J. Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids. Nucleic Acids Res. 2008, 36, 6363–6371.
[21]
Radzio, J.; Sluis-Cremer, N. Efavirenz accelerates HIV-1 reverse transcriptase ribonuclease H cleavage, leading to diminished zidovudine excision. Mol. Pharmacol. 2008, 73, 601–606.
[22]
von Wyl, V.; Ehteshami, M.; Demeter, L.M.; Burgisser, P.; Nijhuis, M.; Symons, J.; Yerly, S.; Boni, J.; Klimkait, T.; Schuurman, R.; et al. HIV-1 reverse transcriptase connection domain mutations: Dynamics of emergence and implications for success of combination antiretroviral therapy. Clin. Infect. Dis. 2010, 51, 620–628.
[23]
Price, H.; Asboe, D.; Pozniak, A.; Gazzard, B.; Fearnhill, E.; Pillay, D.; Dunn, D. Positive and negative drug selection pressures on the N348I connection domain mutation: New insights from in vivo data. Antivir. Ther. 2010, 15, 203–211.
[24]
Menendez-Arias, L.; Betancor, G.; Matamoros, T. HIV-1 reverse transcriptase connection subdomain mutations involved in resistance to approved non-nucleoside inhibitors. Antiviral. Res. 2011, 92, 139–149, doi:10.1016/j.antiviral.2011.08.020.
[25]
Kensch, O.; Restle, T.; W¨ohrl, B.M.; Goody, R.S.; Steinhoff, H.J. Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling. J. Mol. Biol. 2000, 301, 1029–1039, doi:10.1006/jmbi.2000.3998.
Seckler, J.M.; Barkley, M.D.; Wintrode, P.L. Allosteric suppression of HIV-1 reverse transcriptase structural dynamics upon inhibitor binding. Biophys. J. 2011, 100, 144–153.
[28]
Decha, P.; Intharathep, P.; Udommaneethanakit, T.; Sompornpisut, P.; Hannongbua, S.; Wolschann, P.; Parasuk, V. Theoretical studies on the molecular basis of HIV-1RT/NNRTIs interactions. J. Enzyme Inhib. Med. Chem. 2011, 26, 29–36, doi:10.3109/14756360903563393.
[29]
Ivetac, A.; McCammon, J.A. Elucidating the inhibition mechanism of HIV-1 non-nucleoside reverse transcriptase inhibitors through multicopy molecular dynamics simulations. J. Mol. Biol. 2009, 388, 644–658, doi:10.1016/j.jmb.2009.03.037.
[30]
Treesuwan, W.; Hannongbua, S. Bridge water mediates nevirapine binding to wild type and Y181C HIV-1 reverse transcriptase-evidence from molecular dynamics simulations and MM-PBSA calculations. J. Mol. Graph. Model. 2009, 27, 921–929, doi:10.1016/j.jmgm.2009.02.007.
[31]
Eyal, E.; Yang, L.; Bahar, I. Anisotropic network model: Systematic evaluation and a new web interface. Bioinformatics 2006, 22, 2619–2627, doi:10.1093/bioinformatics/btl448.
[32]
Bakan, A.; Meireles, L.M.; Bahar, I. ProDy: Protein dynamics inferred from theory and experiments. Bioinformatics 2011, 27, 1575–1577.
[33]
Temiz, N.A.; Bahar, I. Inhibitor binding alters the directions of domain motions in HIV-1 reverse transcriptase. Proteins 2002, 49, 61–70, doi:10.1002/prot.10183.
[34]
Humphrey, W.; Dalke, A.; Schulten, K. VMD Visual Molecular Dynamics. J. Mol. Graph. 1996, 14, 33–38, doi:10.1016/0263-7855(96)00018-5.
Jorgensen, W.L.; Chandrasekhar, J.; Madura, J.D.; Impey, R.W.; Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926–935.
[37]
Sch¨uttelkopf, A.W.; van Aalten, D.M.F. PRODRG: A tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 2004, 60, 1355–1363.
Wang, J.; Wolf, R.M.; Caldwell, J.W.; Kollman, P.A.; Case, D.A. Development and testing of a general Amber force field. J. Comput. Chem. 2004, 25, 1157–1174, doi:10.1002/jcc.20035.
[41]
Duan, Y.; Wu., C.; Chowdhury., S.; Lee, M.C.; Xiong, G.; Zhang., W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; et al. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 2003, 24, 1999–2012.
Ryckaert, J.P.; Ciccotti, G.; Berendsen, H.J.C. Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327–341, doi:10.1016/0021-9991(77)90098-5.
[45]
Berendsen, H.J.C.; Postma, J.P.M.; van Gunsteren, W.F.; DiNola, A.; Haak, J.R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684–3690.
[46]
Zasada, S.J.; Coveney, P.V. Virtualizing access to scientific applications with the application hosting environment. Comput. Phys. Comm. 2009, 180, 2513–2525.
[47]
Keller, P.A.; Leach, S.P.; Luu, T.T.; Titmuss, S.J.; Griffith, R. Development of computational and graphical tools for analysis of movement and flexibility in large molecules. J. Mol. Graph. Model. 2000, 18, 235–41, 299, doi:10.1016/S1093-3263(00)00028-0.
[48]
Esnouf, R.M.; Ren, J.; Garman, E.F.; Somers, D.O.; Ross, C.K.; Jones, E.Y.; Stammers, D.K.; Stuart, D.I. Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration. Acta Crystallogr. D 1998, 54, 938–953, doi:10.1107/S0907444998004284.
[49]
Chamberlain, P.P.; Ren, J.; Nichols, C.E.; Douglas, L.; Lennerstrand, J.; Larder, B.A.; Stuart, D.I.; Stammers, D.K. Crystal structures of Zidovudine-or Lamivudine-resistant human immunodeficiency virus type 1 reverse transcriptases containing mutations at codons 41, 184, and 21. J. Virol. 2002, 76, 10015–10019.
[50]
Ren, J.; Nichols, C.E.; Chamberlain, P.P.; Weaver, K.L.; Short, S.A.; Stammers, D.K. Crystal structures of HIV-1 reverse transcriptases mutated at codons 100, 106 and 108 and mechanisms of resistance to non-nucleoside inhibitors. J. Mol. Biol. 2004, 336, 569–578, doi:10.1016/j.jmb.2003.12.055.
[51]
Ren, J.; Esnouf, R.; Garman, E.; Somers, D.; Ross, C.; Kirby, I.; Keeling, J.; Darby, G.; Jones, Y.; Stuart, D. High resolution structures of HIV-1 RT from four RT-inhibitor complexes. Nat. Struct. Biol. 1995, 2, 293–302, doi:10.1038/nsb0495-293.
[52]
Ren, J.; Nichols, C.E.; Stamp, A.; Chamberlain, P.P.; Ferris, R.; Weaver, K.L.; Short, S.A.; Stammers, D.K. Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138. FEBS J. 2006, 273, 3850–3860.
[53]
Ren, J.; Milton, J.; Weaver, K.L.; Short, S.A.; Stuart, D.I.; Stammers, D.K. Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase. Structure 2000, 8, 1089–1094, doi:10.1016/S0969-2126(00)00513-X.
[54]
Ren, J.; Nichols, C.; Bird, L.; Chamberlain, P.; Weaver, K.; Short, S.; Stuart, D.I.; Stammers, D.K. Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation non-nucleoside inhibitors. J. Mol. Biol. 2001, 312, 795–805, doi:10.1006/jmbi.2001.4988.
[55]
Bakan, A.; Bahar, I. The intrinsic dynamics of enzymes plays a dominant role in determining the structural changes induced upon inhibitor binding. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 14349–14354, doi:10.1073/pnas.0904214106.
[56]
Loya, S.; Gao, H.Q.; Avidan, O.; Boyer, P.L.; Hughes, S.H.; Hizi, A. Subunit-specific mutagenesis of the cysteine 280 residue of the reverse transcriptase of human immunodeficiency virus type 1: Effects on sensitivity to a specific inhibitor of the RNase H activity. J. Virol. 1997, 71, 5668–5672.
[57]
Purohit, V.; Balakrishnan, M.; Kim, B.; Bambara, R.A. Evidence that HIV-1 reverse transcriptase employs the DNA 3’ end-directed primary/secondary RNase H cleavage mechanism during synthesis and strand transfer. J. Biol. Chem. 2005, 280, 40534–40543.
[58]
Hanson, M.N.; Balakrishnan, M.; Roques, B.P.; Bambara, R.A. Effects of donor and acceptor RNA structures on the mechanism of strand transfer by HIV-1 reverse transcriptase. J. Mol. Biol. 2005, 353, 772–787, doi:10.1016/j.jmb.2005.08.065.
[59]
Biondi, M.J.; Beilhartz, G.L.; McCormick, S.; Gotte, M. N348I in HIV-1 reverse transcriptase can counteract the nevirapine-mediated bias toward RNase H cleavage during plus-strand initiation. J. Biol. Chem. 2010, 285, 26966–26975.
[60]
Sadiq., K.S.; Wan, S.; Coveney, P.V. Insights into a mutation-assisted lateral drug escape mechanism from the HIV-1 protease active site. Biochemistry 2007, 46, 14865–14877, doi:10.1021/bi700864p.
[61]
Stoica, I.; Sadiq, S.; Coveney, P.V. Rapid and accurate prediction of binding free energies for saquinavir-bound HIV-1 proteases. J. Am. Chem. Soc. 2008, 130, 2639–2648.
[62]
Sadiq, S.K.; Wright, D.W.; Kenway, O.A.; Coveney, P.V. Accurate ensemble molecular dynamics binding free energy ranking of multidrug-resistant HIV-1 proteases. J. Chem. Inf. Model. 2010, 50, 890–905.
[63]
Wright, D.W.; Coveney, P.V. Resolution of discordant HIV-1 protease resistance rankings using molecular dynamics simulations. J. Chem. Inf. Model. 2011, 51, 2636–2649, doi:10.1021/ci200308r.
