Proton Coupling and the Multiscale Kinetic Mechanism of a Peptide Transporter

Proton coupled peptide transporters (POTs) are crucial for the uptake of di- and tri-peptides as well as drug and pro-drug molecules in prokaryotes and eukaryotic cells. We illustrate from multiscale modeling how transmembrane proton flux couples within a POT protein to drive essential steps of the full functional cycle: 1) protonation of a glutamate on transmembrane helix (TM) 7 opens the extracellular gate, allowing ligand entry; 2) inward proton flow induces the cytosolic release of ligand by varying the protonation state of a second conserved glutamate on TM10; 3) proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible via water proton shuttling without the participation of ligand. Our results, for the first time, give direct computational confirmation for the alternating access model of POTs, and point to a quantitative multiscale kinetic picture of the functioning protein mechanism. SIGNIFICANCE Proton-coupled peptide transporters (POTs) utilize transmembrane proton gradient to deliver small peptides and peptide-like drug molecules into cells. Despite extensive biochemical and structural studies, major question regarding protonation-induced shift from inward-facing state to outward-facing state remains obscure. Here, we report direct evidence through multiscale simulations that the extracellular salt bridge controls the outward-open conformational transition of POTs, and how proton influx through POTs couples ligand transport. The computational modeling also suggests a multiscale kinetic mechanism of POTs. ### Competing Interest Statement The authors have declared no competing interest.