Molecular Dynamics Simulations Reveal the Proton:Peptide Coupling Mechanism in the Bacterial Proton-Coupled Oligopeptide Transporter YbgH

摘要

Proton-coupled oligopeptide transporters (POTs) couple the inward movement of di- or tripeptides with the inward movement of protons. Experimentally, it has been shown that virtually all di- and tripeptides are recognized as substrates, which suggests that it is the backbone of the peptide that determines substrate affinity and specificity. We have previously shown that a conserved E1XXE2R motif is involved in the binding of the proton. Although the proposed protonation site is in close proximity to the peptide binding site, the mechanism by which the POTs couple protonation to peptide binding is not understood. Here, we have performed molecular dynamics simulations on the crystal structure of Escherichia coli POT YbgH in the absence and presence of a proton on the consensus E2 (Glu21) and on both states in the absence and presence of a dipeptide. We observe that the highly conserved Lys118 is able to interact with Glu21 when Glu21 is not protonated but with the dipeptide C-terminus when Glu21 is protonated. Thus, Lys118 provides YbgH with a coupling mechanism sensor that ensures detection of protonation and peptide binding. Furthermore, we observe that the dipeptide initially interacts only with Glu391, with the rest of the peptide being flexible, and becomes stabilized upon interaction with Lys118. This suggests that the peptide binding is a two-step procedure and that the transition from the first to the second step depends upon protonation of Glu21. Finally, we observe occluded conformations of YbgH during the simulations. Most strikingly, in YbgH devoid of peptide, the highly conserved residues Tyr26 and Arg29 interact with Glu391, overlapping with the space that would otherwise be occupied by a bound peptide. This intramolecular substrate mimicry may explain how the apo transporter returns back into the outside-facing conformation.