Insight into the role of Mg²⁺ in hammerhead ribozyme catalysis from X-ray crystallography and molecular dynamics simulation

Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg²⁺ ion) and 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 Mg²⁺ ion bridging the A9 and scissile phosphates. In the reactant state, the Mg²⁺ spends significant time closely associated with the 2′OH of G8 but remains fairly distant from the leaving group Q_5′ position. In the early TS mimic simulation, where the nucteophilic O_2′ and leaving group O_5′ are equidistant from the phosphorus, the Mg²⁺ 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 replaced 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 Mg²⁺ ion in catalysis that is consistent with both crystallographic and biochemical data.