Insight into the Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from X-ray Crystallography and Molecular Dynamics Simulation

Abstract

Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg 2+ ion), early and late transition state mimics are presented based on a recently reported crystal structure of a full-length hammerhead RNA. The simulation results support a catalytically active conformation with a Mg 2+ ion bridging the A9 and scissile phosphates. In the reactant state, the Mg 2+ spends significant time closely associated with the 2′OH of G8, but remains fairly distant from the leaving group O 5′ position. In the early TS mimic simulation, where the nucleophilic O 2′ and leaving group O 5′ are equidistant from the phosphorus, the Mg 2+ ion remains tightly coordinated to the 2′OH of G8, but is positioned closer to the O 5′ leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg 2+ ion undergoes a transition whereby the coordination with the 2′OH of G8 is replace by the leaving group O 5′ that has developed significant charge. At the same time, the 2′OH of G8 forms a hydrogen bond with the leaving group O 5′ and is positioned to act as a general acid catalyst. This work represents the first reported simulations of the full-length hammerhead structure and TS mimics, and provides direct evidence for the possible role of a bridging Mg 2+ ion in catalysis that is consistent with both crystallographic and biochemical data.The hammerhead ribozyme [1] is an archetype system to study RNA catalysis. [2,3] A detailed understanding of the hammerhead mechanism provides insight into the inner workings of more complex cellular catalytic RNA machinery such as the ribosome, and ultimately may aid the rational design of new medical therapies [4] and biotechnology. [5,6] Despite a tremendous amount of experimental and theoretical effort, [1,2,7,8] the details of the hammerhead ribozyme mechanism have been elusive. In particular, one of the main puzzles involves the apparent inconsistency between the interpretation of thio effect experiments [9,10] and mutational data [8] with available crystallographic structural information of the minimal hammerhead sequence. [11,12,13] Results from the biochemical experiments suggest that a pH-dependent conformational change, inconsistent with crystallographic data, [11,12,13] must precede or be concomitant with the catalytic chemical step. This includes a possible metal ion bridge between the A9 and scissile phosphates that in previous crystal structures were ~20 Å apart. Moreover, the function of the 2′OH group of G8 remains unclear. A stable Mg 2+ ion bridge between the A9 and scissile phosphates is formed in the catalytically active conformationThe simulation results support a catalytic role for a Mg 2+ ion bridging the A9 and scissile phosphates. In the simulations with a bridging Mg 2+ ion, the average distance between the A9 and scissile phosphates remain within the crystallographic value of 4.3 Å, whereas in the absence of Mg 2+ this key contact between stems I and II drifts to over 7 ...

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

“…38,41 Recent crystallographic studies of the extended HHR (eHHR) have characterized the ground state active site architecture 73 and its solvent structure, 74 including the binding mode of a presumed catalytically active divalent metal ion in the active site. These findings, together with molecular simulation studies, 75–79 have reconciled a longstanding controversy between structural and biochemical studies for this system. 80 …”

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

“…These tertiary interactions result in a higher population of functionally folded ribozymes, leading to higher observed rates at significantly lower divalent metal concentrations in vitro, as well as activity in vivo (Khvorova et al 2003). New structures of constructs based on native HHRz's (2GOZ, 2OEU, 2QUS) largely satisfy predictions based on previous mutations and also provide new hypotheses concerning players in the HHRz mechanism (Martick and Scott 2006;Lee et al 2007;Chi et al 2008;Martick et al 2008). The conformation of the native HHRz in crystalforming conditions is significantly different from that of the truncated HHRz.…”

Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

“…29 Only very recently has a solvent structure of the fulllength hammerhead ribozyme become available24 with divalent Mn 2+ positions unambiguously determined. This was made possible by crystallization of the more electronrich Mn 2+ ions that possess a distinct X-ray absorption K edge at 1.896 Å.…”

“…Several fairly short (approximately 1.2 ns or less) molecular dynamics simulations, based on the minimal sequence hammerhead structures, have been reported,25-28 but to date there has only been communication of preliminary simulation results of the full-length hammerhead sequence with the active site in an in-line conformation, and some speculation as to the divalent metal binding mode. 29 The purpose of the present work is to explore in detail the role of Mg 2+ ions in stabilizing the active conformation and proceeding to the transition state, based on the new crystallographic structure of the full-length hammerhead ribozyme with a divalent metal bound near the active site. Toward this end, we have performed a series of eight 12-ns classical molecular dynamics simulations and two 1-ns combined quantum mechanical/ molecular mechanical (QM/MM) simulations with different metal ion occupations at various stages along the chemical reaction coordinate.…”