Dynamical characterization and multiple unbinding paths of two PreQ1 ligands in one pocket.

Riboswitches naturally regulate gene expression in bacteria by binding to specific small molecules. Class 1 preQ₁ 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 preQ₁-I_I riboswitch aptamer to two preQ₁ ligands in one binding pocket. Decomposition of binding free energy suggested that preQ₁ ligands at α and β sites interact with four nucleotides (G5, C17, C18, and A30) and two nucleotides (A12 and C31), respectively. Mg²⁺ ions play a crucial role in both stabilizing the binding pocket and facilitating ligand binding. The flexible preQ₁ 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 preQ₁ ligand at the α site unbinds through two orthogonal pathways, which are dependent on whether or not a β site preQ₁ ligand is present. One of the two preQ₁ ligands has been identified in the binding pocket, which will aid to identify the second preQ₁ Ligand. Our work provides new information for designing robust ligands. [ABSTRACT FROM AUTHOR]