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Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

ABSTRACT: Despite numerous structural and biochemical investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed a total of approximately 300 ns of molecular dynamics (MD) simulations. Our simulations predict improved global stability for an in vitro selected "gain of function" mutation, which is validated by native gel electrophoretic mobility shift assay. We observe that active site nucleobases and water molecules stabilize a geometry favorable to catalysis through a dynamic hydrogen bonding network. Simulations in which A38 is unprotonated show its N1 move into close proximity of the active site 2'-OH, indicating that A38 may act as a general base during cleavage, a role that has generally been discounted due to the longer distances observed in crystal structures involving inactivating substrate analogs. By contrast, simulations in which N1 of A38 is protonated place N1 in close proximity to the 5'-oxygen leaving group, which supports the proposal that A38 serves as a general acid. In analogy to protein enzymes, we discuss a plausible mechanism in which A38 acts bifunctionally and shuttles a proton directly from the 2'-OH to the 5'-oxygen. Furthermore, our simulations suggest an important role for protonation of N1 of A38 in promoting a favorable geometry similar to that observed in transition-state analog crystal structures, and support previously proposed roles of A38, G8, and long residency water molecules in transition-state stabilization.

SUBMITTER: Ditzler MA

PROVIDER: S-EPMC2661834 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

Ditzler Mark A MA Sponer Jirí J Walter Nils G NG

RNA (New York, N.Y.) 20090217 4

Despite numerous structural and biochemical investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed a total of approximately 300 ns of molecular dynamics (MD) simulations. Our simulations predict improved global stability for an in vitro selected "gain of function" mutation, which is validated by native gel electrophoretic mobility shift assay. We ob ...[more]

PMID: 19223444

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Project description:Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, "D5", which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all of its adenine residues labeled with (13)C and (15)N and utilizing Raman difference spectroscopy, we identify the conformationally sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg(2+) binding was explored by analyzing the Raman difference spectra for [D5 + Mg(2+)] minus [D5 no Mg(2+)], for D5 unlabeled, or D5 labeled with (13)C/(15)N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg(2+) binding at the N7 position. In the A-labeled spectra we attribute a Raman differential near 1450 cm(-1) and changes of intensity at 1296 cm(-1) to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg(2+) binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm(-1) upon magnesium binding is due to a "tightening up" (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine-labeled D5, small changes in the adenine backbone bond signatures in the 810-830 cm(-1) region suggest that small conformational changes occur in the tetraloop and bulge regions upon binding of Mg(2+). The PO(2)(-) stretching vibration, near 1100 cm(-1), from the nonbridging phosphate groups, probes the effect of Mg(2+)-hydrate inner-sphere interactions that cause an upshift. In turn, the upshift is modulated by the presence of monovalent cations since in the presence of Na(+) and Li(+) the upshift is 23 +/- 2 cm(-1) while in the presence of K(+) and Cs(+) it is 13 +/- 3 cm(-1), a finding that correlates with the differences in hydration radii. These subtle differences in electrostatic interactions may be related to observed variations in catalytic activity. For a reconstructed ribozyme comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans, cleavage of spliced exon substrates in the presence of magnesium and K(+) or Cs(+) is more efficient than that in the presence of magnesium with Na(+) or Li(+).

Dataset Information

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

Publications

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

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