Ribozyme Catalysis with a Twist: Active State of the Twister Ribozyme in Solution Predicted from Molecular Simulation

Abstract

We present results from molecular dynamics simulations and free energy calculations of the twister ribozyme at different stages along the reaction path in order to gain insight into its mechanism. Results, together with recent biochemical experiments, provide support for a mechanism involving general acid catalysis by a conserved adenine residue in the active site. Although adenine has been previously implicated as a general acid acting through the N1 position in other ribozymes such as the hairpin and VS ribozymes, in the twister ribozyme there may be - a twist. Biochemical experiments suggest that general acid catalysis may occur through the N3 position, which has never before been implicated in this role; however, there currently lacks a detailed structural model for the active state of the twister ribozyme in solution that is consistent with these and other experiments. Simulations in a crystalline environment reported here are consistent with X-ray crystallographic data, and suggest that crystal packing contacts trap the RNA in an inactive conformation with U-1 in an extruded state that is incompatible with an in-line attack to the scissile phosphate. Simulations in solution, on the other hand, reveal this region to be dynamic and able to adopt a conformation where U-1 is stacked with G33. In this state, the nucleophile is in line with the scissile phosphate, and the N1 position of G33 and N3 position of A1 are poised to act as general base and acid, respectively, as supported by mutational experiments. Free energy calculations further predict the electrostatic environment causes a shift of the microscopic pKa at the N3 position of A1 toward neutrality by approximately 5 pKa units. These results offer a unified interpretation of a broad range of currently available experimental data that points to a novel mode of general acid catalysis through the N3 position of an adenine nucleobase, thus expanding the repertoire of known mechanistic strategies employed by small nucleolytic ribozymes.

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

“…Based on molecular modelling simulations, the authors observed that rotation of U-1 towards A34 brought the nucleobase to the expected position underneath G33 with the O2′ atom presenting the required in-line orientation for the nucleophilic attack. Based on restrained molecular dynamics (MD) simulations, Gaines and York explored different conformational states of U-1 in Oryza sativa twister ribozyme and confirmed the existence of the two previously described conformations, termed as extruded and stacked [22].…”

“…Lilley and co-workers suggested that the origin of this shift must be found in the local environment, since the six-membered ring of A1 lies between three phosphate groups [23]. This pKa shift was predicted by Gaines and York from computational simulations that rendered a variation of the pKa at the N3 position of A1 towards neutrality by approximately five units [22].…”

“…Thus, a relatively rigid network of hydrogen bonding, electrostatic and π-stacking interactions around the self-cleavage site was then proposed as being important in either the stabilization of the geometry and catalysis. The study of Hammes-Schiffer and co-workers [24], published just after the work of Gaines and York [22], suggested a quite different mechanism for the Oryza sativa and env22 twister ribozymes.…”

Pressure modulates the self-cleavage step of the hairpin ribozyme

“…To complement our experiments, we explored the self-cleavage step by simulating the activated precursor (AP) state33 being the HpRz structure closest to the transition state (see below for more details), using large-scale replica-exchange molecular dynamics simulations at HHP conditions. Fortunately, we could build on a rich available literature on force field molecular dynamics simulations of RNA or ribozymes at ambient conditions as documented, for instance, in refs 34, 35, 36 among many other works. Importantly, recently improved force fields3738 have enabled fairly reliable such simulations up to long time scales39.…”

Molecular Dynamics Study of Twister Ribozyme: Role of Mg2+ Ions and the Hydrogen-Bonding Network in the Active Site

“…All of these MD trajectories were analyzed to investigate the role of the Mg 2+ ions in twister ribozyme and to examine possible candidates for the general base and acid in the self-cleavage mechanism. During completion of this work, a computational study of the O. sativa twister ribozyme, which has a sequence different from that of the env22 twister ribozyme studied herein, was published; 31 a brief comparison of these two studies is presented in the Conclusions.…”