The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H(+) in the active site, which acts as the general acid, and a partially hydrated Mg(2+) ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with a distinct position and coordination of the catalytically important active site Mg(2+) ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal mol(-1), indicating that the specific position of the Mg(2+) ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2'-OH) nucleophile and the nucleophilic attack of the resulting U-1(2'-O(-)) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimate the pKa of the U-1(2'-OH) group to range from 8.8 to 11.2, suggesting that it is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg(2+) ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg(2+) ion, facilitate deprotonation and activation of the 2'-OH nucleophile.

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacky University tr 17 listopadu 12 771 46 Olomouc Czech Republic

Zobrazit více v PubMed

Doudna JA, Lorsch JR. Nat Struct Mol Biol. 2005;12:395–402. PubMed

Nakano S, Chadalavada DM, Bevilacqua PC. Science. 2000;287:1493–1497. PubMed

Perrotta AT, Shih IH, Been MD. Science. 1999;286:123–126. PubMed

Fedor MJ, Williamson JR. Nat Rev Mol Cell Biol. 2005;6:399–412. PubMed

Bevilacqua PC, Brown TS, Nakano S, Yajima R. Biopolymers. 2004;73:90–109. PubMed

Gong B, Chen JH, Chase E, Chadalavada DM, Yajima R, Golden BL, Bevilacqua PC, Carey PR. J Am Chem Soc. 2007;129:13335–13342. PubMed

Lilley DM. Trends Biochem Sci. 2003;28:495–501. PubMed

Ke AL, Zhou KH, Ding F, Cate JHD, Doudna JA. Nature. 2004;429:201–205. PubMed

Krasovska MV, Sefcikova J, Spackova N, Sponer J, Walter NG. Journal of Molecular Biology. 2005;351:731–748. PubMed

Krasovska MV, Sefcikova J, Spackova N, Sponer J, Walter NG. J Mol Biol. 2005;351:731–748. PubMed

Krasovska MV, Sefcikova J, Reblova K, Schneider B, Walter NG, Sponer J. Biophys J. 2006;91:626–638. PubMed PMC

Banas P, Rulisek L, Hanosova V, Svozil D, Walter NG, Sponer J, Otyepka M. J Phys Chem B. 2008;112:11177–11187. PubMed PMC

Das SR, Piccirilli JA. Nat Chem Biol. 2005;1:45–52. PubMed

Chen JH, Yajima R, Chadalavada DM, Chase E, Bevilacqua PC, Golden BL. Biochemistry. 2010;49:6508–6518. PubMed

Ferre-D'Amare AR, Zhou K, Doudna JA. Nature. 1998;395:567–574. PubMed

Chadalavada DM, Knudsen SM, Nakano S, Bevilacqua PC. J Mol Biol. 2000;301:349–367. PubMed

Golden BL. Biochemistry. 2011;50:9424–9433. PubMed PMC

Veeraraghavan N, Ganguly A, Chen JH, Bevilacqua PC, Hammes-Schiffer S, Golden BL. Biochemistry. 2011;50:2672–2682. PubMed PMC

Ganguly A, Bevilacqua PC, Hammes-Schiffer S. J Phys Chem Lett. 2011;2:2906–2911. PubMed PMC

Ganguly A, Thaplyal P, Rosta E, Bevilacqua PC, Hammes-Schiffer S. Journal of the American Chemical Society. 2014;136:1483–1496. PubMed PMC

Mlynsky V, Banas P, Walter NG, Sponer J, Otyepka M. J Phys Chem B. 2011;115:13911–13924. PubMed PMC

Mlynsky V, Banas P, Sponer J, van der Kamp MW, Mulholland AJ, Otyepka M. Journal of Chemical Theory and Computation. 2014;10:1608–1622. PubMed

D TA, Case DA, Cheatham TE, III, Simmerling CL, Wang J, Duke RE, Luo R, W RC, Zhang W, Merz KM, Roberts B, Hayik S, Roitberg A, Seabra G, Swails J, AW G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wolf RM, Liu J, Wu X, B SR, Steinbrecher T, Gohlke H, Cai Q, Ye X, Wang J, Hsieh MJ, Cui G, R DR, Mathews DH, Seetin MG, Salomon-Ferrer R, Sagui C, Babin V, Luchko T, G S, Kovalenko A, Kollman PA. AMBER 12. University of California; San Francisco: 2012.

Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA. Journal of the American Chemical Society. 1995;117:5179–5197.

Wang JM, Cieplak P, Kollman PA. Journal of Computational Chemistry. 2000;21:1049–1074.

Perez A, Marchan I, Svozil D, Sponer J, Cheatham TE, Laughton CA, Orozco M. Biophys J. 2007;92:3817–3829. PubMed PMC

Banas P, Hollas D, Zgarbova M, Jurecka P, Orozco M, Cheatham TE, Sponer J, Otyepka M. Journal of Chemical Theory and Computation. 2010;6:3836–3849. PubMed PMC

Zgarbova M, Otyepka M, Sponer J, Mladek A, Banas P, Cheatham TE, 3rd, Jurecka P. Journal of Chemical Theory and Computation. 2011;7:2886–2902. PubMed PMC

Sklenovsky P, Florova P, Banas P, Reblova K, Lankas F, Otyepka M, Sponer J. Journal of Chemical Theory and Computation. 2011;7:2963–2980. PubMed

Banas P, Sklenovsky P, Wedekind JE, Sponer J, Otyepka M. Journal of Physical Chemistry B. 2012;116:12721–12734. PubMed PMC

Kuhrova P, Banas P, Best RB, Sponer J, Otyepka M. Journal of Chemical Theory and Computation. 2013;9:2115–2125. PubMed

Besseova I, Banas P, Kuhrova P, Kosinova P, Otyepka M, Sponer J. Journal of Physical Chemistry B. 2012;116:9899–9916. PubMed

Cheatham TE, Case DA. Biopolymers. 2013;99:969–977. PubMed PMC

Svensson M, Humbel S, Froese RDJ, Matsubara T, Sieber S, Morokuma K. J Phys Chem. 1996;100:19357–19363.

Frisch MJ, T GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, et al. Gaussian 09. Gaussian, Inc.; Wallingford CT: 2009.

Lynch BJ, Fast PL, Harris M, Truhlar DG. Journal of Physical Chemistry A. 2000;104:4811–4815.

Lynch BJ, Truhlar DG. Journal of Physical Chemistry A. 2001;105:2936–2941.

Banas P, Jurecka P, Walter NG, Sponer J, Otyepka M. Methods. 2009;49:202–216. PubMed PMC

Otyepka M, Banas P, Magistrato A, Carloni P, Damborsky J. Proteins. 2008;70:707–717. PubMed

Dahm SC, Derrick WB, Uhlenbeck OC. Biochemistry. 1993;32:13040–13045. PubMed

Sponer J, Banas P, Jurecka P, Zgarbova M, Kuhrova P, Havrila M, Krepl M, Stadlbauer P, Otyepka M. Journal of Physical Chemistry Letters. 2014;5:1771–1782. PubMed

Gresh N, Sponer JE, Spackova N, Leszczynski J, Sponer J. J Phys Chem B. 2003;107:8669–8681.

Ditzler MA, Otyepka M, Sponer J, Walter NG. Accounts of Chemical Research. 2010;43:40–47. PubMed PMC

Shih IH, Been MD. Biochemistry. 2000;39:9055–9066. PubMed

Thaplyal P, Ganguly A, Golden BL, Hammes-Schiffer S, Bevilacqua PC. Biochemistry. 2013;52:6499–6514. PubMed PMC

Warshel A, Sharma PK, Kato M, Xiang Y, Liu HB, Olsson MHM. Chem Rev. 2006;106:3210–3235. PubMed

Hertel KJ, Herschlag D, Uhlenbeck OC. Biochemistry. 1994;33:3374–3385. PubMed

Young KJ, Gill F, Grasby JA. Nucleic Acids Res. 1997;25:3760–3766. PubMed PMC

McCarthy TJ, Plog MA, Floy SA, Jansen JA, Soukup JK, Soukup GA. Chem Biol. 2005;12:1221–1226. PubMed

Wilson TJ, McLeod AC, Lilley DM. EMBO J. 2007;26:2489–2500. PubMed PMC

Izatt RM, Hansen LD, Rytting JH, Christensen JJ. J Am Chem Soc. 1965;87:2760–2761. PubMed

Usher DA, Richardson DI, Jr, Oakenfull DG. J Am Chem Soc. 1970;92:4699–4712. PubMed

Jarvinen P, Oivanen M, Lonnberg H. J Org Chem. 1991;56:5396–5401.

Li YF, Breaker RR. J Am Chem Soc. 1999;121:5364–5372.

Lyne PD, Karplus M. J Am Chem Soc. 2000;122:166–167.

Davies JE, Doltsinis NL, Kirby AJ, Roussev CD, Sprik M. J Am Chem Soc. 2002;124:6594–6599. PubMed

Acharya S, Foldesi A, Chattopadhyaya J. J Org Chem. 2003;68:1906–1910. PubMed

Nakano S, Bevilacqua PC. J Am Chem Soc. 2001;123:11333–11334. PubMed

Sripathi KN, Tay WW, Banas P, Otyepka M, Sponer J, Walter NG. RNA. 2014;20:1–17. PubMed PMC

An intricate balance of hydrogen bonding, ion atmosphere and dynamics facilitates a seamless uracil to cytosine substitution in the U-turn of the neomycin-sensing riboswitch

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

Chemical feasibility of the general acid/base mechanism of glmS ribozyme self-cleavage