Significance: The uptake of nutrients from the environment is an essential process that is achieved in most cells through the use of secondary active transporters. The POT family of proton-coupled peptide transporters are one of the most diverse nutrient uptake systems, recognizing amino acids, peptides, nitrate, and seed-defense compounds. A long-standing question is how this family achieves such ligand diversity. A high-resolution crystal structure combined with multiscale molecular dynamics simulations demonstrate water molecules are able to shuttle protons using a Grotthuss-type mechanism, suggesting a separation of ligand recognition from proton movement. This would have clear advantages for a transporter family that must accommodate chemically diverse ligands while retaining the ability to couple transport to the proton electrochemical gradient.

Despite several years of research, the ion exchange mechanisms in chloride/proton antiporters and many other coupled transporters are not yet understood at the molecular level. Here, we present a novel approach to kinetic modeling and apply it to ion exchange in ClC-ec1. Our multiscale kinetic model is developed by (1) calculating the state-to-state rate coefficients with reactive and polarizable molecular dynamics simulations, (2) optimizing these rates in a global kinetic network, and (3) predicting new electrophysiological results. The model shows that the robust Cl:H exchange ratio (2.2:1) can indeed arise from kinetic coupling without large protein conformational changes, indicating a possible facile evolutionary connection to chloride channels. The E148 amino acid residue is shown to couple chloride and proton transport through protonation-dependent blockage of the central anion binding site and an anion-dependent pK_a value, which influences proton transport. The results demonstrate how an ensemble of different exchange pathways, as opposed to a single series of transitions, culminates in the macroscopic observables of the antiporter, such as transport rates, chloride/proton stoichiometry, and pH dependence.