ykkC-I Riboswitch Function Requires an Interplay Between Metal Triad and Cationic Ligand: Insights into Metal Ion-Induced Allostery from Molecular Dynamics Simulations

Riboswitches are non-coding mRNA regions that regulate gene expression by sensing small molecules. While most riboswitches turn off gene expression, guanidine-I family riboswitches enhance gene expression. Although crystal structures provide insights into the structural basis for guanidinium ion (Gdm+) sensing by the guanidine-I riboswitch aptamer (GRA) domain, the mechanistic interplay between ligand and metal ion binding, RNA conformational changes, and regulatory function remains poorly understood. Using molecular dynamics simulations, we explore the combined effects of the positively charged Gdm+, Mg2+, and K+ observed in close proximity in the experimental crystal structure on the GRA structural dynamics. Our simulations reveal that the binding pocket frequently transitions between ligand bound-like and unbound-like states in the absence of divalent ions, while Mg2+ stabilizes a bound-like RNA conformation. Furthermore, both Mg2+ and Gdm+ facilitate K+ positioning near the binding pocket. As a result, Mg2+, Gdm+, and K+ synergistically increase the structural rigidity of the GRA domain, particularly the P2–P3 junction and the 3′ end near the terminator stem. This enhances localized interactions that pull the P1a and P3 domains together to make the transcriptional control region available for expression. Our proposed mechanism is fully consistent with experimental structural and biochemical (including isothermal titration calorimetry and structure-guided mutagenesis) data and rationalizes how the unique triad of ions work together to influence the conformational dynamics of the aptamer domain and riboswitch function. This information can guide future synthetic riboswitch design and the identification of novel therapeutic targets beyond static structural information.