Uncovering the Mechanism of the Proton-Coupled Fluoride Transport in the CLC^F Antiporter

Abstract

Fluoride is a natural antibiotic abundantly present in the environment and, in micromolar concentrations, is able to inhibit enzymes necessary for bacteria to survive. However, as is the case with many antibiotics, bacteria have evolved resistance methods, including through the use of recently discovered membrane proteins. One such protein is the CLCF F-/H+ antiporter protein, a member of the CLC superfamily of anion-transport proteins, most notably known for their ability to transport chloride ions. While it possesses many similarities to the other CLC proteins, it also differs in several key ways, and though previous studies have examined this F- transporter, many questions are still left unanswered. To reveal details of the transport mechanism used by CLCF, we have employed molecular dynamics simulations and umbrella sampling calculations. Our results have led to several discoveries, including the mechanism of proton import and how it is able to aid in the fluoride export. Additionally, we have determined the role of the previously identified residues Glu118, Glu318, Met79, and Tyr396. This work is among the first computational studies of the CLCF F-/H+ antiporter and is the first to propose a mechanism which couples both the proton and anion transport.

Fluoride Ion Binding and Translocation in the CLCF Fluoride/Proton Antiporter: Molecular Insights from Combined Quantum-Mechanical/Molecular-Mechanical Modeling

“…This can be rationalized by fact that the rotation of the E118 side chain is reversible and that the negatively charged carboxylate and fluoride anions can compete with each other for the external binding site. 5 Just like Mills and Torabifard, 22 we found that the unrestrained F cen ions in WT1 diffused quickly away from their initial positions in the binding sites after the simulation started, being 8 Å away within 110 ps for protein monomer A and 690 ps for monomer B, and eventually moved into the bulk solution (Figure S3 in the Supporting Information). In the other models, loosely restrained F cen stayed near the central binding site.…”

“…(ii) Our simulations followed the original windmill mechanism, where fluoride and E118 side chain compete for the external binding site, whereas the modified windmill mechanism without such competitions was pursued in ref. 22 The repulsions between the negatively charged fluoride and carboxylate of E118 in our simulations will almost certainly lead to a higher barrier. Because two different mechanisms were investigated in this work and in ref, 22 the results in one study do not necessarily rule out the other.…”

“…This barrier is notably higher than the 2−6 kcal/mol reported by Mills and Torabifard. 22 The discrepancy is likely due to two factors: (i) we have adopted a QM/MM description, while MM potentials were employed in ref. 22 The stronger interactions between fluoride and its surroundings by QM can lead to higher barriers.…”

“…22 The discrepancy is likely due to two factors: (i) we have adopted a QM/MM description, while MM potentials were employed in ref. 22 The stronger interactions between fluoride and its surroundings by QM can lead to higher barriers. (ii) Our simulations followed the original windmill mechanism, where fluoride and E118 side chain compete for the external binding site, whereas the modified windmill mechanism without such competitions was pursued in ref.…”

“…More recently, Mills and Torabifard 22 conducted dynamic simulations at the MM level and observed that F cen left spontaneously in a very short time. Consequently, the authors concluded that there should be only one fluoride ion along the anion pathway at any time.…”