Proton-Coupled Transition between Outward-Facing and Inward-Facing States in the Uracil Transporter

Abstract

The bacterial nucleobase-ascorbate transporters are proton-driven membrane symporters. The solved structure of the bacterial uracil transporter, which demonstrates an inward-facing open state, is spatially organized into a core domain and a gate domain. Two glutamate residues (Glu241 and Glu290) anchor the substrate uracil in its binding site at the interface between the two domains. However, the outward-facing conformation and the conformational transition between the inward-facing and the outward-facing states are completely unknown. Furthermore, and mechanistically more important, how protons drive this transition remains also elusive. In the present study, we apply molecular dynamics simulations and free energy perturbation (FEP) to address some of these questions. The results of equilibrium simulations show that the empty transporter adopts an outward-facing open conformation through a rigid-body movement of TM5 and TM12. Uracil binding and protonation of Glu241 lead to an occluded state of the transporter. Deprotonation of Glu241 and protonation of His245 (as a result of proton transfer between these side chains) induce the displacement of TM12 from the core domain, exposing the bound uracil to the cytoplasm. Our FEP calculations show that the energy in the uracil bound state with protonated His245 is ∼25.9 kcal/mol lower than that with protonated Glu241, suggesting a favorable proton transfer from Glu241 to His245. Lastly, the release of the proton from protonated His245 in the uracil-bound state causes a large separation of TM12 from the core domain, thus allowing the substrate to move out of its binding site and into the cytoplasm. Based on our simulations, we propose that TM5 and TM12 play the role of extracellular and intracellular gates, respectively, and that proton transfer between Glu241 and His245 regulates the conformational transition between the inward-facing and outward-facing states.