Operation Mechanism of CLCF Fluoride/Proton Antiporter

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Project Summary/Abstract Widely applied to prevent dental caries, F− ion can inhibit bacterial growth. However, many bacterial strains have evolved to be resistant to F–, utilizing exporters situated in the bacterial cell membranes that quickly reduce the intracellular F– concentration. Our long-term goal is to unlock the molecular details of these F– exporters. Our objective in this proposal is to elucidate the operation mechanism of a prototypical CLCF F–/H+ antiporter from E. casseliflavus, which regulates F– efflux and H+ influx with a 1:1 stoichiometric ratio. Our central hypothesis is that multiple structural and energetic factors, including ionic size, electrostatic interactions, hydrogen-bonding, and anion-H+ coupling, are fine-tuned to determine the F– selection and transport in CLCF. We have formulated the hypothesis based on experimental studies of CLCF by Miller and coworkers and on our and others' previous computational work on the homologous canonical CLC Cl–/H+ antiporters. We will carry a series of steered molecular dynamics and umbrella sampling simulations to answer the questions in three specific aims: (1) How are H+ and anions transported in CLCF-Eca? It has been hypothesized that the H+ gate E118 undergoes rotation, carrying H+ from the extracellular to intracellular solutions and propelling F– through the pore. We also hypothesize that F– passes more easily than Cl– due to its smaller radius. We will identify the two protein conformations that are currently missing in the transport cycle, and we will quantify the translocation free-energy barriers for both anions. (2) Why is F– permeation decreased when E318, a residue near the anion binding site, is neutralized? The working hypothesis is that neutralizing the negatively charged E318 in the E318Q and E318A mutants reduces the electrostatic forces that displace F– from the binding site. We will compare the barriers for anion displacements among WT, E318Q, and E318A. (3) What causes the switch in anion selectivity from F– to Cl– in the E118Q and E118A mutants, in which the H+ pathway is abolished? We hypothesize that H+ concentration gradients drive H+ into the anion pore to protonate E318 and that the H+ sharing between F– (but not Cl–) and E318, which have similar pKa values, traps F– in the pore, disrupting the transport cycle. We will simulate water wires formation and subsequent H+ migration. We will estimate free-energy barriers for anion permeation in E118Q and E118Q/E318A; we predict that the double mutant reduces permeation for both anions but retains the F–-over-Cl– selectivity, which can be experimentally tested. The study is innovative because it will (a) shift the current research paradigm for CLCF by including insights from the computational perspective and (b) use novel adaptive-partitioning quantum- mechanics/molecular-mechanics algorithms to simulate explicit H+ transport. The research is significant in that it will (i) provide critical insights into F– resistance in the oral microbial community, (ii) deepen our understanding of the homologous canonical CLC Cl–/H+ and other transport proteins, and (iii) enhance the research environment at CU-Denver Downtown Campus and promote undergraduate student research.