Simulation-Guided Conformational Space Exploration to Assess Reactive Conformations of a Ribozyme.

Self-splicing ribozymes are small ribonucleic acid (RNA) enzymes that catalyze their own cleavage through a transphosphoesterification reaction. While this process is involved in some specific steps of viral RNA replication and splicing, it is also of importance in the context of the (putative) first autocatalytic RNA-based systems that could have preceded the emergence of modern life. The uncatalyzed phosphoester bond formation is thermodynamically very unfavorable, and many experimental studies have focused on understanding the molecular features of catalysis in these ribozymes. However, chemical reaction paths are short-lived and not easily characterized by experimental approaches, so molecular simulation approaches appear as an ideal tool to unveil the molecular details of the reaction. Here, we focus on the model hairpin ribozyme. We show that identifying a relevant initial conformation for reactivity studies, which is frequently overlooked in mixed quantum-classical studies that predominantly concentrate on the chemical reaction itself, can be highly challenging. These challenges stem from limitations in both available experimental structures (which are chemically altered to prevent self-cleavage) and the accuracy of force fields, together with the necessity for comprehensive sampling. We show that molecular dynamics simulations, combined with extensive conformational phase space exploration with Hamiltonian replica-exchange simulations, enable us to characterize the relevant conformational basins of the minimal hairpin ribozyme in the ligated state prior to self-cleavage. We find that what is usually considered a canonical reactive conformation with active site geometries and hydrogen-bond patterns that are optimal for the addition-elimination reaction with general acid/general base catalysis is metastable and only marginally populated. The thermodynamically stable conformation appears to be consistent with the expectations of a mechanism that does not require the direct participation of ribozyme residues in the reaction. While these observations may suffer from forcefield inaccuracies, all investigated forcefields lead to the same conclusions upon proper sampling, contrasting with previous investigations on shorter timescales suggesting that at least one reparametrization of the Amber99 forcefield allowed to stabilize aligned active site conformations. Our study demonstrates that identifying the most pertinent reactant state conformation holds equal importance alongside the accurate determination of the thermodynamics and kinetics of the chemical steps of the reaction.

Consistent Picture of Phosphate-Divalent Cation Binding from Models with Implicit and Explicit Electronic Polarization.

The binding of divalent cations to the ubiquitous phosphate group is essential for a number of key biological processes, such as DNA compaction, RNA folding, or interactions of some proteins with membranes. Yet, probing their binding sites, modes, and associated binding free energy is a challenge for both experiments and simulations. In simulations, standard force fields strongly overestimate the interaction between phosphate groups and divalent cations. Here, we examine how different strategies to include electronic polarization effects in force fields─implicitly, through the use of scaled charges or pair-specific Lennard-Jones parameters, or explicitly, with the polarizable force fields Drude and AMOEBA─capture the interactions of a model phosphate compound, dimethyl phosphate, with calcium and magnesium divalent cations. We show that both implicit and explicit approaches, when carefully parameterized, are successful in capturing the overall binding free energy and that common trends emerge from the comparison of different simulation approaches. Overall, the binding is very moderate, slightly weaker for Ca2+ than Mg2+, and the solvent-shared ion pair is slightly more stable than the contact monodentate ion pair. The bidentate ion pair is higher in energy (or even fully unstable for Mg2+). Our results thus suggest practical ways to capture the divalent cations with biomolecular phosphate groups in complex biochemical systems. In particular, the computational efficiency of implicit models makes them ideally suited for large-scale simulations of biological assemblies, with improved accuracy compared to state-of-the-art fixed-charge force fields.