Extensive Molecular Dynamics Simulations Showing That Canonical G8 and Protonated A38H⁺ Forms Are Most Consistent with Crystal Structures of Hairpin Ribozyme

Abstract

The hairpin ribozyme is a prominent member of the group of small catalytic RNAs (RNA enzymes or ribozymes) because it does not require metal ions to achieve catalysis. Biochemical and structural data have implicated guanine 8 (G8) and adenine 38 (A38) as catalytic participants in cleavage and ligation catalyzed by the hairpin ribozyme, yet their exact role in catalysis remains disputed. To gain insight into dynamics in the active site of a minimal self-cleaving hairpin ribozyme, we have performed extensive classical, explicit-solvent molecular dynamics (MD) simulations on timescales of 50-150 ns. Starting from the available X-ray crystal structures, we investigated the structural impact of the protonation states of G8 and A38, and the inactivating A−1(2′-methoxy) substitution employed in crystallography. Our simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 profoundly distorts the active site. Thus MD simulations do not support a straightforward participation of the deprotonated G8 in catalysis. By comparison, the G8 enol tautomer is structurally well tolerated, causing only local rearrangements in the active site. Furthermore, a protonated A38H + is more consistent with the crystallography data than a canonical A38. The simulations thus support the notion that A38H + is the dominant form in the crystals, grown at pH 6. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the structures of active site and S-turn. Yet, we occasionally also observe formation of a stable A−1(2′-OH)…A38(N1) hydrogen bond, which documents the ability of the ribozyme to form this hydrogen bond, consistent with a potential role of A38 as general base catalyst. The presence of this hydrogen bond is, however, incompatible with the expected in-line attack angle necessary for self-cleavage, requiring a rapid transition of the deprotonated 2′-oxyanion to a position more favorable for in-line attack after proton transfer from A−1(2′-OH) to A38(N1). The simulations revealed a potential force field artifact, occasional but irreversible formation of 'ladder-like', underwound A-RNA structure in one of the external helices. Although it does not affect the catalytic center of the hairpin ribozyme, further studies are under way to better assess possible influence of such force field behavior on long RNA simulations.

Large-Scale Analysis of 48 DNA and 48 RNA Tetranucleotides Studied by 1 μs Explicit-Solvent Molecular Dynamics Simulations

“…4 Since then, work by a number of groups has identified the need to reparameterize the terms that describe the glycosidic bond dihedrals in order to overcome poor reproduction of NMR observables for nucleosides, 5 and to improve the modeling of A-RNA regions, 6, 7 for which the formation of artefactual “ladder-like” structures had been reported. 8, 9 Thanks to these efforts, there are now several alternative parameter sets for the glycosidic bond dihedrals for RNA that are available 5, 7, 10 as well as at least one such parameter set for DNA. 11 More recently, reparameterizations of other backbone dihedrals have been proposed.…”

Computer Folding of RNA Tetraloops: Identification of Key Force Field Deficiencies

“…This metric is significantly more robust compared to RMSD. Notably, in the case of 10-mer TL, even the spurious ladder-like structure 32, 45 is ~3.8 Å in RMSD from the native conformation. 29 The εRMSD is able to clearly distinguish this spurious state as a non-native conformation.…”

“…32, 40, 44 In 2010 we identified a similar serious problem in RNA simulations, namely the formation of ladder-like structures in canonical A-form helices. 45 Subsequently we derived a reparameterization of the glycosidic torsion χ OL3 32, 38 which supressed the ladder-like structures and became a part of the contemporary standard AMBER force field. Suppression of the RNA ladder-like structure required the DNA and RNA AMBER force fields to be separated, due to incompatible requirements on the χ dihedral potentials in DNA and RNA.…”

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

“…In the latter case, simulations suggested that a deprotonated G8 − is incompatible with the active site architecture observed in the crystal structures. 38 However, subsequent QM/MM calculations have indicated that the active site containing a deprotonated G8 − is sufficiently reactive to overcome the thermodynamic penalty arising from the rarity of the guanine protonation state, so that the mechanism involving G8 − can be considered a plausible reaction pathway for the hairpin ribozyme. 39 In other words, these mechanisms do not necessarily require a stable architecture of a rare active site ionization state that would be easily accessed by classical force field simulations.…”