Couplings between Local and Global Conformational Changes in Proton-Coupled Oligopeptide Transporters

Abstract

Here we report on an extensive set of equilibrium and nonequilibrium all-atom molecular dynamics (MD) simulations of a bacterial proton-coupled oligopeptide transporter (POT), namely GkPOT, in an explicit membrane/water environment. Using several microseconds of unbiased MD trajectories, we have characterized both the local and global conformational dynamics of the transporter upon the proton and/or substrate binding, within the simulation timescales. Our results reveal a distinct behavior for local conformational dynamics in the absence and presence of the proton at the putative proton binding site. Particularly, we find that the substrate binding conformation is drastically different in the two conditions; the substrate binds to the protein in an either lateral or vertical manner, in the presence or absence of the proton, respectively. This behavior is consistently observed in multiple sets of independent simulations for different substrates. On the other hand, we do not observe any statistically significant distinctive behavior in terms of the global conformational changes under different simulation conditions. On the other hand, the linear regression analysis of quantities associated with the global conformational fluctuations provides evidence for a mechanism involving the concerted motion of the transmembrane helices, consistent with the rocker-switch mechanism proposed for major facilitator superfamily transporters. Employing a novel biasing scheme based on extrapolating the global rotational fluctuations of the transmembrane helices, we have reconstructed the inward- to outward-facing conformational transition of the GkPOT transporter in the presence and absence of the substrate/proton. Unlike the unbiased equilibrium simulations, a strong correlation is observed between the local and global conformational changes observed in our nonequilibrium simulations. These observations provide evidence for a strong coupling between the protein global conformational changes and local binding site conformational changes. However, these couplings are not observed, in a statistically sound manner, when only the unbiased equilibrium simulations are considered, even on the microsecond timescale. Our results, therefore, call into question the implicit assumption behind MD studies that use short simulations to speculate on long-timescale behavior of the membrane transporters, which often function on timescales much longer than those currently accessible to unbiased MD.