Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Role of Protonation States of G8 and A38 Nucleobases in Structure Stabilization and Catalysis of the Hairpin Ribozyme
The hairpin ribozyme is a prominent member of the group of small RNA enzymes (ribozymes) because it does not require metal ions to achieve catalysis of the reversible, site-specific cleavage of its RNA substrate (1). Guanine 8 (G8) and adenine 38 (A38) were identified by biochemical and structural experiments as the key participants involved in cleavage. Despite broad efforts, their exact roles in catalysis remains disputed.(2) We have carried out classical molecular dynamics (MD) simulations (3,4) in explicit solvent on 50-150 ns timescales with various protonation states of G8 and A38. The MD simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 distorts the active site. The G8 enol tautomer is structurally well tolerated, causing only local rearrangements in the active site. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the active site. The Protonated A38H+ is more consistent with the crystallography data.(5) MD simulations support the idea that A38H+ is the dominant form in the crystals, grown at pH 6. This finding is also in agreement with recent NAIM experimental data (6). The MD simulations were also used to find geometries of potential reactive states bearing G8 and A38 of various protonation states in the active site. These geometries were further analyzed by hybrid quantum-mechanical/molecular mechanical (QM/MM) methods to evaluate the energy along the reaction pathway in order to find the most probable reaction mechanism. The DFT MPW1K/6-31+G(d,p) method was used for the QM region and the parm99 force field for MM region, while the communication between these both layers was realized via electronic embedding.(7,8) The quality of our DFT method was tested against reference CCSD(T)/CBS calculations. The mean unassigned error of the DFT method was found to be below 1.0 kcal·mol-1. The calculated activation barriers are in good agreement with experimental data (20-21 kcal·mol-1) for the systems with canonical G8 and both canonical/protonated forms of A38/A38H+ (19.6 kcal·mol-1 and 20.1 kcal·mol-1, respectively). The initial nucleophile attack of the A-1(2’-OH) group on the scissile phosphate is the rate-limiting step along the reaction path. The G8-enol tautomer increases the overall reaction barrier by 4.7 kcal·mol-1. On the other hand, our preliminary data indicate that the presence of an unprotonated G8- (together with A38H+) seems to significantly decrease the activation barrier. Protonated A38H+ does not significantly affect the overall activation barrier (20.1 kcal·mol-1), but decreases the activation barrier of the exocyclic cleavage step by 7.7 kcal·mol-1. In the reaction path where a protonated A38H+ acts as the general acid, the product is 9.5 kcal·mol-1 higher in energy compared to the product in the reaction where a nonbridging oxygen of the scissile phosphate acts as proton donor and A38H+ is not directly involved in cleavage chemistry.
This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (grant IAA400040802), by the Grant Agency of the Czech Republic (grants 203/09/H046, 203/09/1476, P208/11/1822 and P301/11/P558), by Ministry of Youth, Sport and Education of Czech Republic (grants LC512 and MSM6198959216), by Student Project PrF_2010_025 of Palacky University. This work was supported also by the Operational Program Research and Development for Innovations - European Social Fund (CZ.1.05/2.1.00/03.0058) and NIH grant 2R01 GM062357 to NGW.
1Regional Centre of Advanced Technologies and Materials