A cooperative effect of ligands, Mg²⁺ ions, and the U6C mutation on the structural dynamics of the SAM-Ⅵ Riboswitch

SAM riboswitches, the most abundant type of riboswitch, are classified into eight classes. SAM-VI differs from all other known SAM riboswitch classes by adopting a unique fold and ligand pocket. To elucidate the cooperative structural dynamic effects of ligands, Mg²⁺, and the U6C mutation on SAM-VI, we carried out extensive MD simulations totaling 128 μs. Free energy decomposition identified four key nucleotides (U8, G9, C32, and G33) and suggested a strategy that incorporates entropic contributions for novel ligand design. Approximately eight inner-shell Mg²⁺ ions were predicted, two of which (M3 and M'2) bridge two secondary structures (linking L3-S1 and L2-L1-S2, respectively). In the U6C mutation, M4 and M'8 form inner-shell coordination with the nucleobase of U35, connecting L3 and S3. Bridging Mg²⁺ ions directly alter the RNA secondary structure, and ligand coordination drives additional structural refinement. Collectively, Mg²⁺ ions, ligands, and the U6C mutation exert a hierarchical influence on RNA flexibility: Mg²⁺ and SAM synergistically stabilize the riboswitch, whereas U6C disrupts this stability by elevating regional fluctuation. This work defines the molecular basis of cooperative regulation in SAM-VI, establishing a framework for understanding riboswitch dynamic control and rational ligand design.