Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Non-Sequential Phosphate Pairs

Abstract

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg2+, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg2+ in folded conformations of RNA have been studied extensively, however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) simulations are used to capture the energetics and atomic-level details of Mg2+-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg2+, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg2+ with two or more non-sequential phosphate groups. Notably, the present results indicate a push-pull mechanism where the Mg2+-RNA interactions actually lead to destabilization of specific non-sequential phosphate-phosphate interactions while other interactions are stabilized, a balance that facilitates the folding and stabilization of Twister including formation hydrogen bonds associated with the tertiary structure. The present study establishes a better understanding of how Mg2+ ion-interactions contribute to RNA structural properties and stability.\n\nStatement of SignificanceRNAs are biologically and therapeutically of great emerging interest such that it is critical to understand how RNA molecules fold into complex structures. While experiments yield information on the stabilization of RNA by ions they are limited in the atomic-level insights they can provide. A combination of enhanced sampling methods is applied to explore the compact intermediate states of RNA and their interactions with Mg2+ ions. Results reveal a picture of how Mg2+ overall stabilizes short phosphate-phosphate interactions thereby facilitating the stabilization of RNA, though doing so by both the stabilization and destabilization of specific interactions. The applied method will be applicable to exploring the impact of divalent ions on the conformational heterogeneity of a range of macromolecules.