The Mechanism of the Hammerhead Ribozyme RNA Cleavage Reaction: Determining Optimized Free Energy Reaction Paths from QM/MM Simulations
EMSL Project ID
47701
Abstract
Ribonucleic acids (RNA) play a central role in the transmission of genetic information. However, the identification of additional non-coding roles of RNA polymers continues to grow. An important class of these behaviors is the discovery of the catalytic function of RNA molecules. A well studied example is the hammerhead ribozyme (HHR), which facilitates the self cleavage of RNA during rolling circle replication of plant viroids. We are requesting support to utilize the Chinook supercomputer to develop a well validated, theoretically based reaction mechanism for the self-cleavage reaction of HHR. Our goal is to produce a minimum free energy path from the reactant (RS) to the product (PS) state that is consistent with all the existing biochemical and structural data for this well studied ribozyme, is based on reliable 1st principle electronic structure calculations, and incorporates as complete an exploration of the possible reaction mechanisms as feasible. Recently the publication of a high resolution X-ray structure of an inactive analog of the S.mansoni HHR reactive state has reconciled many discrepancies between biochemical observations and structural studies of the enzyme-substrate complex. However, many questions concerning the detailed chemical mechanism of this reaction cannot be answered from structural data from an inactive enzyme complex. There are additional experimental probes, pH variation, thio modification rescue experiments, mutagenesis and chemical modification experiments. As yet these diverse data have not been correlated by a consistent reaction mechanism calculated using 1st principle methods that will support prediction for the chemical diversity of the experimental species involved. Theoretical interpretations have been reported. However, these calculations are based on QM/MM models in which the quantum region was treated with semiempirical methods and in addition certain as yet only weakly justified assumptions about the reaction mechanism. For example, a specific general acid/base pair (as yet uncertain from observation) was selected for the calculation and initial proton transfer from the assumed nuclophile to the assumed general base is taken as the initial structure. As arguably the best understood ribozyme (best structural and biochemical information available) this self-cleavage reaction provides an promising target for complete free energy analysis. Our objective is to identify a full reaction path using: high level 1st principle simulation techniques (calculations at the B3LYP level); implementation of sampling methods (metadynamics and temperature-accelerated molecular dynamics) to identify candidate structures and reaction paths: nudged elastic band and string methods to optimize reaction paths; and finite temperature free energy simulations allowing the full structural relaxation of enzyme substrate complex along the reaction path. Since similar residue motifs have been identified in other ribozymes the understanding of the HHR system would support the successful application of theoretical interpretations of this mechanism would lead to a more insight into the chemistry of other ribozyme and to a deeper understanding of other phosphoryl transfer reactions in protein based enzymes. The team assembled to carry out this project contains experts in the development and application of 1st principle methods (Valiev, Elaesser, Weare, and Pirojsirikul (graduate student)) and experts in ribozyme chemistry (Muller, Elaesser).
Project Details
Project type
Exploratory Research
Start Date
2012-12-03
End Date
2013-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Elsasser BM, G Fels, and JH Weare. 2013. "QM/MM Simulation (B3LYP) of the RNase A Cleavage-Transesterification Reaction Supports a Triester AN+DN Associative Mechanism with an O2´ H Internal Proton Transfer." Journal of the American Chemical Society 136(3):927-936. doi:10.1021/ja406122c
Elsasser BM, I Schoenen, and G Fels. 2013. "Comparative Theoretical Study of the Ring-Opening Polymerization of Caprolactam vs Caprolactone Using QM/MM Methods." ACS Catalysis 3(6):1397-1405. doi:10.1021/cs3008297