Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 48

4. HHR USES SPECIFIC CATION-BINDING MODES TO
REACH ITS CATALYTICALLY ACTIVE CONFORMATION
Simulations summarized in Sections 2 and 3 suggested that the diva-
lent metal ion can serve to stabilize active in-line conformations and interact
with key residues to facilitate acid/base catalysis. However, it is known that
under high non-physiological salt concentrations, the HHR and other
ribozymes remain catalytically proficient. 130-132 Hence, a new question
arises whether divalent metal ion occupancy in the active site is strictly nec-
essary or just a sufficient condition to achieve efficient catalysis that might be
affected by other cations and solvent components.
As already mentioned, the modeling of ions in highly charged systems
such as RNA affords tremendous challenges with regard to simulation time-
scales. The reason is that the equilibration and sampling times of mass trans-
port/diffusion limited processes (such as ion migration) and subsequent
macromolecular structural relaxation are notoriously slow. 115
In this section, we summarize the results of a series of five 300-ns sim-
ulations of the eHHR, in both the reactant ground state and activated pre-
cursor state, in order to explore the relationship between cation-binding
modes and active in-line conformation. 133 The results indicate that the
HHR has an electronegative cation-recruiting pocket in the active site that,
upon threshold occupancy, stabilizes catalytically active in-line attack con-
formations via specific cation-binding patterns. These results provide a first
glimpse into a possibly common feature that might be exploited by other
ribozymes: the engineering of electrostatically pre-organized active sites that
are able to recruit solvent components to facilitate catalysis.
4.1. Results
MD simulations were set up to explore the metal ion-binding modes in
eHHR and their relationship to structure and catalysis. Specifically, we con-
sider the “C-site” (see also Section 2 ) derived from a recent joint crystallo-
graphic/molecular simulation study 74 of the solvent structure of the eHHR
that indicates a Mn 2 þ ion is coordinated to the O2P atom of the A9 phos-
phate and the N7 atom of G10.1, and the “B-site” inferred from thio/rescue
effects 62,63 and also predicted from molecular simulations 75,76 where a diva-
lent metal ion bridges A9:O2P and the scissile phosphate (C1.1:O2P). Sim-
ulations, in the absence of divalent metal ions, were also performed since
HHR activity can be recovered by high concentrations of monovalent