The human APOBEC3G protein (A3G) is a single-stranded DNA deaminase that inhibits the replication of retrotransposons and retroviruses, including HIV-1. Atomic details of A3G’s catalytic mechanism have started to emerge, as the structure of its catalytic domain (A3Gctd) has been revealed by NMR and X-ray crystallography. The NMR and crystal structures are similar overall; however, differences are apparent for β2 strand (β2) and loops close to the catalytic site. To add some insight into these differences and to better characterize A3Gctd dynamics, we calculated its free energy profile by using the Generalized-Born surface area (GBSA) method accompanied with a molecular dynamics simulation. The GBSA method yielded an enthalpy term for A3Gctd’s free energy, and we developed a new method that takes into account the distribution of the protein’s dihedral angles to calculate its entropy term. The structure solved by NMR was found to have a lower energy than that of the crystal structure, suggesting that this conformation is dominant in solution. In addition, β2-loop-β2’ configuration was stable throughout a 20-ns molecular dynamics (MD) simulation. This finding suggests that in solution A3Gctd is not likely to adopt the continuous β2 strand configuration present in the APOBEC2 crystal structure. In the NMR structure, the solvent water accessibility of the catalytic Zn 2+ was limited throughout the 20-ns MD simulation. This result explains previous observations in which A3G did not bind or catalyze single cytosine nucleotide, even when at excessive concentrations.
References
[1]
Chiu, Y.L.; Greene, W.C. The APOBEC3 cytidine deaminases: An innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu. Rev. Immunol. 2008, 26, 317–353, doi:10.1146/annurev.immunol.26.021607.090350.
[2]
Goila-Gaur, R.; Strebel, K. HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology 2008, 5, 51, doi:10.1186/1742-4690-5-51.
[3]
Malim, M.H.; Emerman, M. HIV-1 accessory proteins—Ensuring viral survival in a hostile environment. Cell Host Microbe 2008, 3, 388–398, doi:10.1016/j.chom.2008.04.008.
[4]
Sheehy, A.M.; Gaddis, N.C.; Choim, J.D.; Malim, M.H. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002, 418, 646–650, doi:10.1038/nature00939.
[5]
Lecossier, D.; Bouchonnet, F.; Clavel, F.; Hance, A.J. Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 2003, 300, 1112–1112, doi:10.1126/science.1083338.
Mangeat, B.; Turelli, P.; Caron, G.; Friedli, M.; Perrin, L.; Trono, D. Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 2003, 424, 99–103, doi:10.1038/nature01709.
[8]
Betts, L.; Xiang, S.; Short, S.A.; Wolfenden, R.; Carter, C.W., Jr. Cytidine deaminase. The 2.3 angstrom crystal structure of an enzyme: Transition-state analog complex. J. Mol. Biol. 1994, 235, 635–656, doi:10.1006/jmbi.1994.1018.
[9]
Carter, C.W., Jr. The nucleoside deaminases for cytidine and adenosine: Structure, transition state stabilization, mechanism, and evolution. Biochimie 1995, 77, 92–98, doi:10.1016/0300-9084(96)88110-7.
[10]
Xiang, S.; Short, S.A.; Wolfenden, R.; Carter, C.W., Jr. Transition-state selectivity for a single hydroxyl group during catalysis by cytidine deaminase. Biochemistry 1995, 34, 4516–4523.
[11]
Chen, K.M.; Harjes, E.; Gross, P.J.; Fahmy, A.; Lu, Y.; Shindo, K.; Harris, R.S.; Matsuo, H. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. Nature 2008, 452, 116–119, doi:10.1038/nature06638.
[12]
Furukawa, A.; Nagata, T.; Matsugami, A.; Habu, Y.; Sugiyama, R.; Hayashi, F.; Kobayashi, N.; Yokoyama, S.; Takaku, H.; Katahira, M. Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G. EMBO J. 2009, 28, 440–451, doi:10.1038/emboj.2008.290.
[13]
Harjes, E.; Gross, P.J.; Chen, K.M.; Lu, Y.; Shindo, K.; Nowarski, R.; Gross, J.D.; Kotler, M.; Harris, R.S.; Matsuo, H. An extended structure of the APOBEC3G catalytic domain suggests a unique holoenzyme model. J. Mol. Biol. 2009, 389, 819–832, doi:10.1016/j.jmb.2009.04.031.
[14]
Shandilya, S.M.; Nalam, M.N.; Nalivaika, E.A.; Gross, P.J.; Valesano, J.C.; Shindo, K.; Li, M.; Munson, M.; Royer, W.E.; Harjes, E.; Kono, T.; Matsuo, H.; Harris, R.S.; Somasundaran, M.; Schiffer, C.A. Crystal structure of the APOBEC3G catalytic domain reveals potential oligomerization interfaces. Structure 2010, 18, 28–38, doi:10.1016/j.str.2009.10.016.
[15]
Holden, L.G.; Prochnow, C.; Chang, Y.P.; Bransteitter, R.; Chelico, L.; Sen, U.; Stevens, R.C.; Goodman, M.F.; Chen, X.S. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications. Nature 2008, 456, 121–124.
[16]
Prochnow, C.; Bransteitter, R.; Klein, M.G.; Goodman, M.F.; Chen, X.S. The APOBEC-2 crystal structure and functional implications for the deamizase AID. Nature 2007, 445, 447–451.
[17]
Autore, F.; Bergeron, J.R.C.; Malim, M.H.; Fraternali, F.; Huthoff, H. Rationalisation of the Differences between APOBEC3G Structures from Crystallography and NMR Studies by Molecular Dynamics Simulations. PLoS One 2010, 5, e11515.
[18]
Still, W.C.; Tempczyk, A.; Hawley, R.C.; Hendrickson, T. Semianalytical treatment of solvation for molecular mechanics and dynamics. J. Am. Chem. Soc. 1990, 112, 6127–6130, doi:10.1021/ja00172a038.
[19]
Schaefer, M.; Karplus, M.A. Comprehensive Analytical treatment of continuum electrostatics. J. Phys. Chem. 1996, 100, 1578–1599, doi:10.1021/jp9521621.
[20]
Onufriev, A.; Bashford, D.; Case, D.A. modification of the generalized born model suitable for macromolecules. J. Phys. Chem. B 2000, 104, 3712–3720.
[21]
Cramer, J.C.; Truhlar, G.D. General parameterized SCF model for free energies of solvation in aqueous solution. J. Am. Chem. Soc. 1991, 113, 8305–8311, doi:10.1021/ja00022a017.
[22]
Hawkins, D.G.; Cramer, J.C.; Truhlar, G.D. Pairwise solute descreening of solute charges from a dielectric medium. Chem. Phys. Lett. 1995, 246, 122–129, doi:10.1016/0009-2614(95)01082-K.
[23]
Hawkins, D.G.; Cramer, J.C.; Truhlar, G.D. Parametrized models of aqueous free energies of solvation based on pairwise descreening of solute atomic charges from a dielectric medium. J. Phys. Chem. 1996, 100, 19824–19839.
[24]
Watanabe, Y.S.; Fukunishi, Y.; Nakamura, H. Generation of a flexible loop structural ensemble and its application to induced-fit structural changes following ligand binding. Biophysics 2006, 2, 1–12, doi:10.2142/biophysics.2.1.
[25]
Watanabe, Y.S.; Kim, J.G.; Fukunishi, Y.; Nakamura, H. Free energy landscapes of small peptides in an implicit solvent model determined by force-biased multicanonical molecular dynamics simulation. Chem. Phys. Lett. 2004, 400, 258–263, doi:10.1016/j.cplett.2004.10.118.
[26]
Engels, M.; Krüger, P.; Jacoby, E.; Wollmer, A. Targetted molecular dynamics simulation of conformational change—Application to the T ？ R transition in insulin. Mol. Simulat. 1993, 10, 291–308, doi:10.1080/08927029308022170.
[27]
Standley, D.M.; Toh, H.; Nakamura, H. ASH structure alignmentpackage: Sensitivity and selectivity in domain classification. BMCBioinformatics 2007, 4, 116.
[28]
Standley, D.M.; Toh, H.; Nakamura, H. GASH: an improved algorithm formazimizing the number of equivalent residues between two protein structures. BMCBioinformatics 2005, 6, 221.
[29]
Fujitani, H.; Matsuura, A.; Sakai, S.; Sato, H.; Tanida, T. High-level ab initio calculations to improve protein backbone dihedral parameters. J. Chem. Theor. Comput. 2009, 5, 1155–1165, doi:10.1021/ct8005437.
[30]
Case, D.A.; Darden, T.A.; Cheatham, T.E., III.; Simmerling, C.L.; Wang, J.; Duke, R.E.; Luo, R.; Merz, K.M.; Wang, B.; Pearlman, D.A.; et al. AMBER 8; University of California: San Francisco, CA, USA, 2004.
[31]
Jorgensen, W.L.; Chandrasekhar, J.; Madura, J.D.; Impey, R.W.; Klein, M.L. Comparison of simple potential functions for simulating lipid water. J. Chem. Phys. 1983, 79, 926–935.
[32]
Rankin, W.T. DPMTA—Distributed Parallel Multipole Tree Algorithm; Duke University: Durham, NC, USA, 2002.
[33]
Fukunishi, Y.; Mikami, Y.; Nakamura, H. The filling potential method: A method for estimating the free energy surface for protein-ligand docking. J. Phys. Chem. B 2003, 107, 13201–13210, doi:10.1021/jp035478e.
[34]
myPresto, version 4.207; Osaka University: Suita, Osaka, Japan. Available online: http://presto.protein.osaka-u.ac.jp/myPresto4/index_e.html (accessed on 7 July 2012).
[35]
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. Comp. Phys. 1997, 23, 327–341.
[36]
Richmond, T.J. Solvent accessible surface area and excluded volume in proteins. Analytical equations for overlapping spheres and implications for the hydrophobic effect. J. Mol. Biol. 1984, 178, 63–89, doi:10.1016/0022-2836(84)90231-6.
[37]
Iwatani, Y.; Takeuchi, H.; Strebel, K.; Levin, J.G. Biochemical acivities of highly purified, catalytically active human APOBEC3G: Correlation with antiviral effect. J. Virol. 2006, 80, 5992–6002, doi:10.1128/JVI.02680-05.
[38]
Krzysiak, T.C.; Jung, J.; Thompson, J.; Baker, D.; Gronenborn, A.M. APOBEC2 is a monomer in solution: implications for APOBEC3G models. Biochemistry 2012, 51, 2008–2017.
