Alternative Base-Pairing and Conformational Sampling in Loop a of the Hairpin Ribozyme

Abstract

The catalytic cycle of the hairpin ribozyme begins with the ‘docking’ between loops A and B and the consideration of this step as the rate limiting step for the nucleolytic catalysis presents an intriguing question. The kinetic rates of docking previously reported by ourselves and others indicate that conformational sampling may be a factor that limits the docking transition rates. To assess conformational sampling in the pre-docking form of the hairpin ribozyme, we have run several explicit solvent Molecular Dynamics (MD) simulations of hairpin ribozyme loop A domain totaling 2.4 µs. We observed three dominant conformers and other minor states identified using hydrogen bonding and base stacking in the loop region. Targeted Molecular Dynamics (TMD) was used to model the pathway between a pair of conformations using CHARMM36/NAMD. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPB/SA) approximation was applied to predict conformational free energy and energy differences between various conformations. MMPB/SA correctly predicted the major conformations to be lower in free energy than the minor conformations. The observation of multiple conformations with different energies seem to underscore the formation of a rugged energy landscape with multiple accessible shallow energy minima for loop A of the hairpin ribozyme, consistent with a possible role for conformational sampling in the observed slow rates of docking.