Energetics of substrate transport in proton-dependent oligopeptide transporters.

The PepT transporter mediates the transport of peptides across biological membranes. Despite advancements in structural biology, including cryogenic electron microscopy structures resolving PepT in different states, the molecular basis of peptide recognition and transport by PepT is not fully elucidated. In this study, we employed molecular dynamics simulations, Markov State Models (MSMs), and Transition Path Theory (TPT) to investigate the transport mechanism of an alanine-alanine peptide (Ala-Ala) through the PepT transporter. Our simulations revealed conformational changes and key intermediate states involved in peptide translocation. We observed that the presence of the Ala-Ala peptide substrate lowers the free energy barriers associated with transition to the inward-facing state. Furthermore, we elucidated the proton transport model and analyzed the pharmacophore features of intermediate states, providing insights for rational drug design. These findings highlight the significance of substrate binding in modulating the conformational dynamics of PepT and identify critical residues that facilitate transport.