Molecular dynamics analysis of Mg²⁺‐dependent cleavage of a pistol ribozyme reveals a fail‐safe secondary ion for catalysis

Abstract

Pistol ribozymes comprise a class of small, self-cleaving RNAs discovered via comparative genomic analysis. Prior work in the field has probed the kinetics of the cleavage reaction, as well as the influence of various metal ion cofactors that accelerate the process. In the current study, we performed unbiased and unconstrained molecular dynamics simulations from two current high-resolution pistol crystal structures, and we analyzed trajectory data within the context of the currently accepted ribozyme mechanistic framework. Root-mean-squared deviations, radial distribution functions, and distributions of nucleophilic angle-of-attack reveal insights into the potential roles of three magnesium ions with respect to catalysis and overall conformational stability of the molecule. A series of simulation trajectories containing in silico mutations reveal the relatively flexible and partially interchangeable roles of two particular magnesium ions within solvated hydrogen-bonding distances from the catalytic center. K E Y W O R D S CHARMM, MD simulations, metal dependent catalysis, molecular dynamics, pistol ribozyme, ribozyme

The role of Na+ in catalysis by the 8–17 DNAzyme

“…A similar combination of strategies has been suggested as a part of the mechanism of the pistol ribozyme. [19][20][21][22] Remarkably, these findings are in agreement with the recently proposed existence of a common structural motif shared by five nucleolytic ribozymes and the 8-17 DNAzyme, named the L-scaffold, which is formed by two key elements: the L-platform and the L-pocket, arranged to enable the occurrence of the catalytic strategies displayed in each case. 23 In the 8-17 DNAzyme, this platform serves to position the conserved guanine that acts as a general base close to the 2′-OH nucleophile and the L-pocket facilitates the metal ion binding site, allowing a G + M + paradigm of catalysis.…”

Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

“…[ 22 , 23 , 53 , 55 ] More precisely, for pistol, the nature of the cationic influencer can be associated most likely with the electrostatic potential of the “second” hydrated Mg 2+ ion that is located near G40, observed in all crystal structures of precatalytic, transition state mimic, and product states. [33] At this point we mention that also several computational studies shed light on the possible influencer activities,[ 56 , 57 , 58 ] one of them especially points out that the Hoogsteen edge of G40 is exposed to solvent in an electronegative pocket, making the O6 position available to (transiently) interact with metal ions (considerably more than other guanine residues) and that this can tune the p K a at the N1 position. [56] In another computational study, the interesting finding was made (by means of classical MD simulations and QM/MM hybrid calculations) that only the model where the protonation of the nucleotide base was according to the canonical state renders reactive conformations of the active site.…”

“… [33] At this point we mention that also several computational studies shed light on the possible influencer activities,[ 56 , 57 , 58 ] one of them especially points out that the Hoogsteen edge of G40 is exposed to solvent in an electronegative pocket, making the O6 position available to (transiently) interact with metal ions (considerably more than other guanine residues) and that this can tune the p K a at the N1 position. [56] In another computational study, the interesting finding was made (by means of classical MD simulations and QM/MM hybrid calculations) that only the model where the protonation of the nucleotide base was according to the canonical state renders reactive conformations of the active site. [57] This was not the case for the models in which guanine G40 was parametrized as deprotonated at the N1 atom (“reversed protonation”).…”

Who stole the proton? Suspect general base guanine found with a smoking gun in the pistol ribozyme

“…Pioneering structural and functional studies by Breaker, 24,25 Micura and Patel, 26–28 Lilley and Wilson, 29 and others, 30 and supported by theoretical studies, 31–33 have made clear that a metal ion plays an important role in the acid step of the reaction, and further established a functional link with the hammerhead ribozyme 31 and the 8–17 DNAzyme, 17 although ultimately their mechanisms are distinct.…”