Evidence for the Role of Active Site Residues in the Hairpin Ribozyme from Molecular Simulations along the Reaction Path

Abstract

The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Residues A38 and G8 have been implicated as playing roles in general acid and base catalysis, respectively. Here we explore the structure and dynamics of key active site residues using more than 1 μs of molecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway. Analysis of results indicates hydrogen bond interactions between the nucleophile and proR nonbridging oxygen are correlated with active inline attack conformations. Further, the simulation results suggest a possible alternative role for G8 to promote inline fitness and facilitate activation of the nucleophile by hydrogen bonding, although this does not necessarily exclude an additional role as a general base. Finally, we suggest that substitution of G8 with N7- or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5′ leaving group position, would provide valuable insight into the specific role of G8 in catalysis.

A GPU-Accelerated Parameter Interpolation Thermodynamic Integration Free Energy Method

“…The “ref 125” charge set uses the protonated adenine nucleobase charges developed in ref 125. The “15%” charge set uniformly polarizes the ff14SB nucleobase charges by a factor of 1.15, that is,…”

Combining Simulations and Solution Experiments as a Paradigm for RNA Force Field Refinement

“…[1][2][3][4][5][6][7][8][9] In the case of RNA folding, a few partly successful atomistic simulations have been reported. [10][11][12][13] However, recent extensive simulations of unstructured oligonucleotides for which converged sampling is affordable have unambiguously shown that current force-field parameters are not accurate enough to reproduce solution experiments.…”

Pressure modulates the self-cleavage step of the hairpin ribozyme

“…To complement our experiments, we explored the self-cleavage step by simulating the activated precursor (AP) state33 being the HpRz structure closest to the transition state (see below for more details), using large-scale replica-exchange molecular dynamics simulations at HHP conditions. Fortunately, we could build on a rich available literature on force field molecular dynamics simulations of RNA or ribozymes at ambient conditions as documented, for instance, in refs 34, 35, 36 among many other works.…”

“…The proposed mechanistic explanation is based on the well-known fact that hydrogen bonding of the reaction site with neighbouring nucleobases has a central role at ambient conditions in establishing the trigonal bipyramidal structure that is essential for the chemical reaction step to occur subsequently303233. Here we find that exactly the same hydrogen bonding interactions that prepare the ribozyme for the self-cleavage reaction to set in get even more enhanced under high-pressure conditions, which we propose to explain at the molecular level the experimentally observed pressure-induced acceleration of the self-cleavage reaction in the compressed HpRz.…”