Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

Abstract

Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg2+ ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg2+ ion or multiple Na+ ions, one of which is also in a bridging coordination pattern. In the case of a single Mg2+ ion bound in the active site, the Mg2+ ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2′). As the reaction proceeds, the Mg2+ ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2′. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis.

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

“…Another Mg 2+ is believed to play the role of activating the 2′-OH of the general acid G8 (green) by migrating from the binding site at N7 of G10.1 (“C-site”) observed crystallographically into a bridging position (“B-site”) with the scissile phosphate, in accord with thio/rescue effect experiments(29, 39, 57). In this bridging position, the Mg 2+ can coordinate the 2′-OH of G8, increasing its acidity, and facilitating proton transfer to the O5′ leaving group in the general acid step of the reaction(8). …”

“…The HHR catalyzes the cleavage transesterification of the RNA sugar-phosphate backbone(8). In the generally accepted acid-base mechanism, the nucleophile (the 2′-hydroxyl of residue C17) is deprotonated by a general base to form an activated precursor that then goes on an inline attack to the adjacent scissile phosphate.…”

Multiscale Methods for Computational RNA Enzymology

“…The computational efficiency of these models allows MD simulations to routinely access μs timescales (Salomon-Ferrer, Götz, Poole, Le Grand, & Walker, 2013; Dror, Dirks, Grossman, Xu, & Shaw, 2012), making it a viable method for capturing large scale conformational changes in catalytic riboswitches (Giambaşu et al, 2010; Giambaşu, Lee, Scott, & York, 2012). Taken together, these developments have provided insight into the condensed phase structure and dynamics of ribozymes both in their pre-cleaved ground state and at various points along a reaction path (T.-S. Lee, Giambaşu, Harris, & York, 2011; T.-S. Lee et al, 2010; T.-S. Lee, Wong, Giambasu, & York, 2013). Of key importance to the understanding of ribozyme function is to understand what conformational event leads to the catalytically active pre-cleaved ground state, and how does the ribozyme environment respond so as to preferentially stabilize high energy transition states and intermediates as the reaction progresses.…”

“…In the case of the hairpin ribozyme, electrostatic effects in the active site (Nam, Gao, & York, 2008a) account for a large part of the observed rate acceleration, and cause a shift of the p K a of an adenine nucleobase which acts as a general acid catalyst to facilitate leaving group departure (Nam, Gao, & York, 2008b). The hammerhead ribozyme, on the other hand, has engineered a highly electronegative active site that can recruit a threshold occupation of cationic charge (T.-S. Lee et al, 2009) (a Mg 2+ ion under physiological conditions, or multiple monovalent cations under high salt conditions) that facilitates formation of an active in-line attack conformation (T.-S. Lee, Wong, et al, 2013; T.-S. Lee et al, 2008), stabilizes accumulating charge in the transition state and increase the acidity of the 2′OH group of a conserved guanine residue in order to facilitate proton transfer to the leaving group (Wong, Lee, & York, 2011). In both the hairpin and hammerhead ribozymes, as well as other ribozymes such as the glmS riboswitch (Klein, Been, & Ferré-D’Amaré, 2007; Viladoms, Scott, & Fedor, 2011) and Varkud satellite ribozyme (T.…”

VR-SCOSMO: A smooth conductor-like screening model with charge-dependent radii for modeling chemical reactions

“…Such studies can help interpret experimental results which are pursuing a deeper understanding of the fundamental principles governing chemical activity and encompass a wide range of archetypes including, but not limited to, reactions occurring within protein environments, [1][2][3] model phosphoryl transfer reactions, [4][5][6][7][8] and studies of RNA catalysis. [9][10][11] The end goal of these studies is frequently to aid in the design of new drugs and therapeutic treatments. [12][13][14] Multiscale modeling approaches including long timescale molecular dynamics simulations to explore fluctuations and conformational changes of biomolecules, combined quantum mechanical/molecular mechanical (QM/MM) simulations to examine deeply embedded reactive chemical events, and implicit solvent calculations of small model reactions are used for this task.…”