Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme

Abstract

The Varkud Satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free energy relationship measurements with molecular dynamics, molecular solvation theory, and ab initio quantum mechanical/molecular mechanical free energy simulations to gain insight into catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfers in the transition state. Further, we provide evidence for a critical Mg 2+ ion in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg 2+ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the VS, hairpin, and hammerhead ribozyme classes.

Self-cleaving ribozymes: substrate specificity and synthetic biology applications

“…In addition, Mg 2+ in the active site appears to interact critically with the scissile phosphate and also activates G638. 117 Formation of the kissing loop and the preservation of the function of A756 and G638 are important considerations for substrate specificity of the cis-acting VS ribozyme. Mutation of A756 to G or G638 to A causes a significant decrease (10 3 -10 4fold) in the rate of cleavage.…”

“…In addition, Mg 2+ in the active site appears to interact critically with the scissile phosphate and also activates G638. 117 …”

Development of Range-Corrected Deep Learning Potentials for Fast, Accurate Quantum Mechanical/molecular Mechanical Simulations of Chemical Reactions in Solution

“…[1][2][3] However, the translation of this capability to the latest and most powerful state-of-the-art and emerging quantum mechanical (QM) methods has not kept pace. 4 These high-level QM methods are extremely important for the study of catalytic mechanisms to guide enzyme design, 5 drug discovery, and precision medicine applications. 6 However, in practice, the computational cost of high-level ab initio quantum mechanical/molecular mechanical (QM/MM) [7][8][9][10] or quantum mechanical force field (QMFF) 11 simulations prohibits their practical use for many important applications.…”

“…To demonstrate the accuracy and transferability of the approach using these efficient tools, we studied the 2'-O-transphosphorylation RNA cleavage reactions 51 (Figure 1). This specific phosphoryl transfer (PT) reaction is essential for living organisms, and has been the focus of many structural and mechanistic computational enzymology studies of nucleic acid enzymes known to catalyze it 5,[52][53][54][55] yielding insight into nucleic acid enzyme design. 56 This approach builds upon our recent work to develop new methods for construction of multi-dimensional free energy surfaces 57 and "quantum mechanical book-ending" methods 58 for alchemical free energy simulations, adding to the arsenal of computational tools for free energy prediction in the AMBER suite of programs.…”

Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis

“…59 LFER and computation support extensive and/or complete proton transfer from the 2′-hydroxy to G638, but only partial proton transfer from A756 to the 5′-O leaving group. 15 Accordingly, the VSrz reflects case 2, above, as it has an active site in which the A756 acid is characterized by a low pK a and the G638 base has a high pK a . Thus, over a relatively broad pL range where Δk/ΔpL = 0, there are offsetting changes in the speciation of different protonation states of the active site that can be incorporated into the interpretation of PI results using the GPW-GB equation.…”