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Insight into the role of Mg(II) in hammerhead ribozyme catalysis from X-ray crystallography and molecular dynamics simulation

Recent Research from Professor Darrin York and his research group.

The hammerhead ribozyme is an archetype system to study RNA catalysis. 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 and biotechnology.

Despite a tremendous amount of experimental and theoretical effort, 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 and mutational data with available crystallographic structural information of the minimal hammerhead sequence. Results from the biochemical experiments suggest that a pH-dependent conformational change, inconsistent with crystallographic data, 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.

Recently, the research groups of Professor Darrin York, scientist Tai-Sung Lee and postdoc Carlos Silva-Lopez of the Department of Chemistry, in collaboration with Professor Bill Scott and his graduate student Monika Martick of the Department of Chemistry, UCSC, have performed molecular simulations that probe the conformational events and metal ion binding that leads to ribozyme catalysis. A series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg(II) ion), early and late transition state mimics are presented based on a recent crystal structure of a full-length hammerhead RNA reported by Martick and Scott (Fig. 1).

Figure 1.  The full-length hammerhead RNA reported by Martick and Scott [Cell 126, 309 (2006)].

Their simulation results support a catalytically active conformation with a Mg(II) ion bridging the A9 and scissile phosphates (Fig 2). In the reactant state, the Mg(II) spends significant time closely associated with the 2’OH of G8, but remains fairly distant from the leaving group O5’ position. In the early TS mimic simulation, where the nucleophilic O2’ and leaving group O5’ are equidistant from the phosphorus, the Mg(II) ion remains tightly coordinated to the 2’OH of G8, but is positioned closer to the O5’ leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg(II) ion undergoes a transition whereby the coordination with the 2’OH of G8 is replace by the leaving group O5’ that has developed significant charge. At the same time, the 2’OH of G8 forms a hydrogen bond with the leaving group O5’ and is positioned to act as a general acid catalyst.

Figure 2. Snapshots of the active site from the early TS mimic (left) and late TS mimic (right) simulations depicting the Mg(II) ion direct coordination (green lines) and key hydrogen bonds and indirect Mg(II) coordination (dotted lines). For clarity, the water molecules are not shown.

This work represents the first reported simulations of the full-length hammerhead structure (Fig 2) and TS mimics, and provides direct evidence for the possible role of a bridging Mg(II) ion in catalysis that is consistent with both crystallographic and biochemical data.  It has been published on-line in J. Chem. Theory Comput.
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