Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from Molecular Simulation

Abstract

Molecular dynamics simulations have been performed to investigate the role of Mg 2+ in the fulllength hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/ MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg 2+ ion. The simulations suggest that Mg 2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg 2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg 2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2′OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2′OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg 2+ ion. The present study provides evidence for the role of Mg 2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

“…7D). The same trend is seen in the in-line fitness parameter (Soukup and Breaker 1999;Lee et al 2008), which includes both the IAA and the O29-P attack distance (Fig. 7D, inset).…”

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

“…High-resolution crystal structures exist for the HDV (Ke et al 2004(Ke et al , 2007, hammerhead (Martick and Scott 2006;Lee et al 2008), glmS (Klein and Ferré-D'Amaré 2006;Cochrane et al 2007), and hairpin ribozymes (Rupert and Ferré-D'Amaré 2001;Rupert et al 2002;Alam et al 2005;Salter et al 2006;Torelli et al 2007;MacElrevey et al 2008;Torelli et al 2008). Based on these structures only the hairpin ribozyme's active site is unambiguously devoid of stably bound metal ions that could potentially participate in catalysis with the possible exception of electrostatic stabilization, which could influence chemistry from outside the active site.…”

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

“…However, our previous study identified an apparent increase in Mg 2+ -binding affinity to HH(S) in mixed solutions with a low dielectric constant [34,35]. The substrate cleavage catalyzed by the hammerhead ribozyme requires the involvement of several Mg 2+ ions, and at least one Mg 2+ ion binds to the site with highly negative electrostatic potential located near the scissile phosphate of the substrate [49]. This mechanism is consistent with the dielectric constant effect that facilitates electrostatic Mg 2+ binding and allows greater enhancement of the turnover activity observed at the low Mg 2+ concentration of 1 mM.…”