Guanidine-II aptamer conformations and ligand binding modes through the lens of molecular simulation.

Regulation of gene expression via riboswitches is a widespread mechanism in bacteria. Here, we investigate ligand binding of a member of the guanidine sensing riboswitch family, the guanidine-II riboswitch (Gd-II). It consists of two stem-loops forming a dimer upon ligand binding. Using extensive molecular dynamics simulations we have identified conformational states corresponding to ligand-bound and unbound states in a monomeric stem-loop of Gd-II and studied the selectivity of this binding. To characterize these states and ligand-dependent conformational changes we applied a combination of dimensionality reduction, clustering, and feature selection methods. In absence of a ligand, the shape of the binding pocket alternates between the conformation observed in presence of guanidinium and a collapsed conformation, which is associated with a deformation of the dimerization interface. Furthermore, the structural features responsible for the ability to discriminate against closely related analogs of guanidine are resolved. Based on these insights, we propose a mechanism that couples ligand binding to aptamer dimerization in the Gd-II system, demonstrating the value of computational methods in the field of nucleic acids research.

Influence of Na+ and Mg2+ ions on RNA structures studied with molecular dynamics simulations.

The structure of ribonucleic acid (RNA) polymers is strongly dependent on the presence of, in particular Mg2+ cations to stabilize structural features. Only in high-resolution X-ray crystallography structures can ions be identified reliably. Here, we perform molecular dynamics simulations of 24 RNA structures with varying ion concentrations. Twelve of the structures were helical and the others complex folded. The aim of the study is to predict ion positions but also to evaluate the impact of different types of ions (Na+ or Mg2+) and the ionic strength on structural stability and variations of RNA. As a general conclusion Mg2+ is found to conserve the experimental structure better than Na+ and, where experimental ion positions are available, they can be reproduced with reasonable accuracy. If a large surplus of ions is present the added electrostatic screening makes prediction of binding-sites less reproducible. Distinct differences in ion-binding between helical and complex folded structures are found. The strength of binding (ΔG‡ for breaking RNA atom-ion interactions) is found to differ between roughly 10 and 26 kJ/mol for the different RNA atoms. Differences in stability between helical and complex folded structures and of the influence of metal ions on either are discussed.

Cooperative binding of bivalent ligands yields new insights into the guanidine-II riboswitch.

Riboswitches are involved in regulating the gene expression in bacteria. They are located within the untranslated regions of bacterial messenger RNA and function as switches by adjusting their shape, depending on the presence or absence of specific ligands. To decipher the fundamental aspects of bacterial gene control, it is therefore important to understand the mechanisms that underlie these conformational switches. To this end, a combination of an experimental binding study, molecular simulations and machine learning has been employed to obtain insights into the conformational changes and structural dynamics of the guanidine-II riboswitch. By exploiting the design of a bivalent ligand, we were able to study ligand binding in the aptamer dimer at the molecular level. Spontaneous ligand-binding events, which are usually difficult to simulate, were observed and the contributing factors are described. These findings were further confirmed by in vivo experiments, where the cooperative binding effects of the bivalent ligands resulted in increased binding affinity compared to the native guanidinium ligand. Beyond ligand binding itself, the simulations revealed a novel, ligand-dependent base-stacking interaction outside of the binding pocket that stabilizes the riboswitch.

Using Dimensionality Reduction to Systematically Expand Conformational Sampling of Intrinsically Disordered Peptides.

One of the approaches to improve our ability to characterize biologically important processes and to map out an underlying free energy landscape is to direct MD simulations to explore molecular conformational phase space faster. Intrinsically disordered systems with shallow free energy landscapes of a huge number of metastable minima pose a particular challenge in this regard. Both characterization of the often ill-defined conformational states as well as the assessment of the degree of convergence of phase space exploration are problematic. We have used a multidimensional scaling-like embedding (sketch-map) to describe the energetically accessible regions of phase space for a peptide fragment of the intrinsically disordered protein α-synuclein. Using sketch-map coordinates from a short initial simulation, we guided additional MD simulations to efficiently expand sampling of the conformational space. The sketch-map projections are very well suited to detect rare but possibly functionally relevant events, metastable intermediates, and transition states in the vast amount of data.