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Abstract

The thiamine pyrophosphate (TPP) riboswitch, located in the mRNA 5'-UTR, consists of an aptamer domain and an expression platform. TPP dissociation from the ligand-binding site in the aptamer domain triggers an allosteric transition that increases the structural flexibility of the P1 helix and activates the expression platform for thiamine biosynthesis. However, the structural mechanism underlying this allosteric transition remains poorly understood. In this study, we investigated this mechanism using molecular simulations and NMR experiments. We employed a generalized-ensemble molecular dynamics simulation to perform extensive conformational sampling and to compute free-energy landscapes of the aptamer domain in the complex and ligand-free states at 300 K. Comparison of the two states revealed that ligand dissociation induces a contraction of the ligand-binding pocket. This structural change increases the distance between the C15 and G51 bases located at the root of the P1 helix, thereby destabilizing base-pair formation and triggering an increase in the conformational flexibility of the P1 helix. Notably, the C15–G51 base pair is located more than 20 Å from the ligand-binding site. Furthermore, the imino-proton exchange rate measured by NMR confirmed the destabilization of the C15–G51 base pair by ligand dissociation. These findings provide a mechanistic explanation for the allosteric transition triggered by ligand dissociation and offer insight into how this process regulates thiamine biosynthesis.