Dynamical characterization and multiple unbinding paths of two PreQ 1 ligands in one pocket.

Riboswitches naturally regulate gene expression in bacteria by binding to specific small molecules. Class 1 preQ 1 riboswitch aptamer is an important model not only for RNA folding but also as a target for designing small molecule antibiotics due to its well-known minimal aptamer domain. Here, we ran a total of 62.4 μs conventional and enhanced-sampling molecular dynamics (MD) simulations to characterize the determinants underlying the binding of the preQ 1 -I I riboswitch aptamer to two preQ 1 ligands in one binding pocket. Decomposition of binding free energy suggested that preQ 1 ligands at α and β sites interact with four nucleotides (G5, C17, C18, and A30) and two nucleotides (A12 and C31), respectively. Mg 2+ ions play a crucial role in both stabilizing the binding pocket and facilitating ligand binding. The flexible preQ 1 ligand at the β site leads to the top of the binding pocket loosening and thus pre-organizes the riboswitch for ligand entry. Enhanced sampling simulations further revealed that the preQ 1 ligand at the α site unbinds through two orthogonal pathways, which are dependent on whether or not a β site preQ 1 ligand is present. One of the two preQ 1 ligands has been identified in the binding pocket, which will aid to identify the second preQ 1 Ligand. Our work provides new information for designing robust ligands.