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. 2024 Mar 6;128(11):2697–2706. doi: 10.1021/acs.jpcb.4c00079

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

Nara L Chon 1, Hai Lin 1,*
PMCID: PMC10962343  PMID: 38447081

Abstract

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CLCF fluoride/proton antiporters move fluoride ions out of bacterial cells, leading to fluoride resistance in these bacteria. However, many details about their operating mechanisms remain unclear. Here, we report a combined quantum-mechanical/molecular-mechanical (QM/MM) study of a CLCF homologue from Enterococci casseliflavus (Eca), in accord with the previously proposed windmill mechanism. Our multiscale modeling sheds light on two critical steps in the transport cycle: (i) the external gating residue E118 pushing a fluoride in the external binding site into the extracellular vestibule and (ii) an incoming fluoride reconquering the external binding site by forcing out E118. Both steps feature competitions for the external binding site between the negatively charged carboxylate of E118 and the fluoride. Remarkably, the displaced E118 by fluoride accepts a proton from the nearby R117, initiating the next transport cycle. We also demonstrate the importance of accurate quantum descriptions of fluoride solvation. Our results provide clues to the mysterious E318 residue near the central binding site, suggesting that the transport activities are unlikely to be disrupted by the glutamate interacting with a well-solvated fluoride at the central binding site. This differs significantly from the structurally similar CLC chloride/proton antiporters, where a fluoride trapped deep in the hydrophobic pore causes the transporter to be locked down. A free-energy barrier of 10–15 kcal/mol was estimated via umbrella sampling for a fluoride ion traveling through the pore to repopulate the external binding site.

1. Introduction

The CLCF proteins are a family of fluoride/proton antiporters that export fluoride driven by proton influx.1−5 By expelling intracellular fluoride, they help many bacteria survive in toxic environments of high fluoride concentrations.6 A lot about these proteins have been learned from the crystal structure of a homologue from Enterococci casseliflavus (CLCF-eca) by Miller and co-workers.5 The protein architecture of CLCF-eca is highly similar to those of the CLC chloride/proton antiporters, e.g., the one from Escherichia coli (CLC-ec1), which has been extensively studied.7−10 Just like CLC-ec1, CLCF-eca comes out as a homodimer, with each monomer possessing its own paths for ion translocations (Figure 1a). Moreover, the external proton gating glutamate residues are in similar positions in CLCF-eca (E118) and CLC-ec1 (E148). Two fluoride binding sites are found in each CLCF-eca monomer in locations comparable to the chloride binding sites in CLCs.11,12 Specifically, the external site is surrounded by backbone amides G116–G119. Located ∼6 Å away, the central site is close to the hydroxyl group of Y396 (equivalent to Y445 in CLC-ec1) and is accessible to intracellular solvent via a wide aqueous vestibule. However, the strictly conserved chloride-coordinating serine in CLC (S107 in CLC-ec1) is missing in CLCF-eca, which possesses instead a conserved methionine, M79, at a roughly equivalent position. Probably, the most surprising finding in CLCF-eca is a glutamate (E318) in close contact (∼3 Å) with the central fluoride ion (Fcen), which has no counterpart in CLC-ec1. It was concluded that E318 must be protonated (pE318) in such an environment, forming a hydrogen bond with Fcen. Although differences in the structures of these two proteins are subtle, their anion transport activities vary notably: first, CLCF-eca selects fluoride over chloride, while such an anion selectivity is reversed in CLC-ec1.5,13 Second, CLCF-eca features a 1:1 anion–proton stoichiometry (i.e., for each discharged fluoride, one proton is imported), while the ratio is 2:1 in CLC-ec1.2,3,5

Figure 1.

Figure 1

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(a) Overall protein architecture of the CLCF homodimer. The protein is shown as helices and loops in green, and the bound fluoride ions as pink spheres. The dashed arrows indicate the directions of ion translocations. (b) Illustration of the windmill mechanism. Key binding-site residues are shown as sticks and fluoride ions as spheres in both the external (ext) and central (cen) binding sites. Color code: H, white; C, cyan; O, red; N, blue; S, yellow; and F, pink. The curved arrow (magenta) indicates the rotation of E118 that leads to alternated “up” and “down” positions.

A windmill mechanism was proposed by Miller and co-workers5 based on the crystal structures of CLCF-eca and a series of mutagenesis experiments and biochemical measurements.2,3,5 Central to the windmill mechanism are the rotatory motions of the E118 side chain, which lead to alternate conformations with ion pathways swapping between intracellular and extracellular sides (Figure 1b). Briefly, the transport cycle starts with a protonated E118 (pE118) in the “up” position. The pE118 rotates to the “down” position and releases the proton into the cytoplasmatic solution, thus completing the inward translocation of the proton. Next, the deprotonated E118 continues to rotate, first pushing Fcen into the intracellular solution and then propelling the external fluoride (Fext) into the extracellular solution. The transport cycle ends with E118 returning to the up position to be reprotonated by an extracellular proton and two fluoride ions being reloaded into the binding sites from the intracellular solution. As such, the windmill mechanism explains the 1:1 anion–proton stoichiometry.

A number of computational studies of CLCF have recently been published. For example, Chon et al.14 analyzed the anion pathways in the wild-type (WT) and two (E118Q and V319G) mutants. Their Poisson–Boltzmann calculations revealed pKa shifts for a number of glutamate and aspartate residues. Notably, pKa of the external gating E118 in the up position was predicted to be ∼7 and further increased to ∼8 upon fluoride occupying the external binding site, suggesting that E118 is ready to be protonated in this position. Furthermore, the buried D32, E170, and E369 located near the putative proton pathways were found to likely be protonated, thus avoiding an excessively negative environment that may trap the proton halfway. The pore size computed using the HOLE15 program suggested that the crystal structures of WT and E118Q are inward-open-outward-occluded, while that of V319G is inward-closed-outward-occluded.

Performing both classical and combined quantum-mechanical/molecular-mechanical (QM/MM)16−18 metadynamic simulations,19 Carloni and co-workers20,21 investigated the proton transfer and release processes. They discovered that the rotation of pE118 from the up to down positions about N–Cα–Cβ–Cγ (χ1) is essentially barrierless. Another important finding is that with pE118 in the down position, it promotes the release of Fcen, which takes the proton from pE318 and leaves as a charge-neutral HF molecule into the intracellular solution. It is expected that pE118 subsequently relays its proton to E318, reprotonating E318. The conclusion of the simultaneous release of the proton and fluoride into the intracellular solution nicely explains the 1:1 stoichiometry of anion/proton exchange.

More recently, Mills and Torabifard22 conducted dynamic simulations at the MM level and observed that Fcen 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. Accordingly, the free-energy profiles were computed through umbrella sampling for one fluoride ion passing through the pore with E118 and E318 in various protonation states. The authors then proposed a modified windmill mechanism, which also explains the 1:1 stoichiometry of anion/proton exchange. However, the modified mechanism necessitates the deprotonated E118 moving from the down to up positions through the anion pore in the absence of Fext. This is in contrast to the original windmill mechanism, where deprotonated E118 drives Fext out of the pore.

While these above theoretical studies have provided valuable insights into the operation mechanisms of CLCF-eca and are generally in line with the experimental results, they disagree with each other in some critical details. For example, the spontaneous departure of Fcen from the binding site in dynamic simulations at the MM level in ref (22) differs notably from the QM/MM simulations in ref (20), where the exit of Fcen was triggered by the arrival of pE118. The discrepancy is probably due to the different methods employed in the modeling as the MM force fields likely underestimate the interactions between a fluoride ion and the protonated carboxylate groups of aspartate and glutamate side chains. The pKa values of hydrogen fluoride (∼3.2), aspartic acid (∼3.9), and glutamic acid (∼4.1) are close to each other, implying that a fluoride ion can form very strong hydrogen bonds or even share a proton (i.e., forming partial covalent bonds), with aspartic and glutamic acids in certain environments.23−26 As revealed in QM/MM simulations of CLC-ec1,14,21,27 such strong interactions between a fluoride ion and the protonated E148 (pE148) have led to the fluoride ion being trapped in the pore, interrupting the transport cycle; this provides a plausible explanation for the experimental observation that fluoride inhibits CLC-ec1 and highlights the importance of accurate descriptions, preferably at the QM level, of the interactions between a fluoride ion and its surroundings.

The investigation by Carloni and co-workers20,21 addressed the proton transport and release into the intracellular solution but did not cover the fluoride translocation. According to the original windmill mechanism, two steps take place after the proton release: (i) the deprotonated E118 propels Fext out of the binding site and into the extracellular solution and (ii) fluoride ions are then reloaded into both binding sites either by directly moving Fext and Fcen each into their respective binding sites or by first converting Fcen into Fext and then reloading Fcen from the intracellular solution. Here, we report a QM/MM study that simulates these two missing steps in the transport cycle. Free-energy barrier is estimated for moving a fluoride ion through the pore via umbrella sampling. The implications of the results to the operating mechanisms of CLCF-eca are discussed. Our findings contribute to a more complete understanding of the fluoride selection, gating, and translocation by CLCF.

2. Methods

2.1. Model Constructions and Equilibrations at the MM level

Beginning with the crystal structures (PDB 6D0J and 6D0N),5 we constructed six (WT1 to WT6) atomistic models of CLCF-eca using the VMD28 program. These models featured various numbers of bound fluoride ions, distinct deprotonation states of E118 and E318, and different (up or down) locations of E118 (see Table S1 in the Supporting Information). In particular, models WT1–WT5 each contained four bound fluoride (two Fext and two Fcen) ions. Each model was built by embedding the protein homodimer in a bilayer of 134 lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or POPC) molecules, followed by solvation by ∼23,000 TIP3P29 water molecules. The membrane was placed in the x–y plane, and the pores of anion translocation were aligned approximately with the z axis. To achieve charge neutrality and a 0.15 M physiological solution concentration, 60 potassium and 62–66 chloride ions (depending on the deprotonation states of E118 and E318) were included. The CAHRMM36m force fields were selected for the proteins, lipids, and ions.30−34 Periodic boundary conditions were applied, and the dimensions of the primary cell containing ∼133,000 atoms were ∼133.7 × 133.7 × 72.0 Å3. After initial minimizations and relaxations, each model was equilibrated under an isothermal–isobaric (NpT) ensemble at 300 K and 1 bar. Equilibrations were run for 20 ns for WT1 and 40 ns for WT2–WT5. The WT6 model contained only two Fcen ions. It was built by removing the two Fext ions from the WT3 model, reneutralizing the overall charge by deleting two randomly selected chloride ions, and re-equilibrating for 40 ns.

All MM calculations in this work were performed using the NAMD (version 2.10) program.35 A cutoff of 12 Å was used for nonbonded short-range interactions with a smoothing function switched on at 11 Å. The particle mesh Ewald36 method was employed for long-range electrostatic interactions. A time step size of 2 fs was used, with the RATTLE37 algorithm invoked to keep the X–H (X = C, N, and O) bonds rigid, and the trajectory was saved every 10 ps. The Fcen ion was not restrained in WT1, but it was loosely restrained to the central binding site in WT2–WT6 by applying harmonic potentials to selected distances between Fcen and nearby atoms.

2.2. QM/MM Simulations

All QM/MM calculations in this work were carried out using a local version of the QMMM38 program, which calls Gaussian1639 for the involved QM calculations and NAMD 2.1035 for the involved MM calculations, synthesizes the QM and MM energies and gradients, and propagates the QM/MM trajectory. The popular density function theory (DFT) model B3LYP40−42 was selected, and all atoms were described by the polarized double-ζ basis set 6-31G(d),43−46 with additional diffuse functions47 assigned to oxygen and fluorine atoms to account for the diffuse electron density for anions. The accuracy of the chosen QM level of theory and basis sets has been well established in our previous studies of the highly similar CLC.48−51 The subtractive definition of the QM/MM energy was adopted, and mechanical embedding was selected for simplicity.17,18 Ordinary hydrogen atoms were employed as link atoms to saturate the dangling bonds at the QM/MM boundary that passed through covalent bonds.52 More details of the QM/MM computations are given in Section S1 in the Supporting Information. The MM subsystem was described by the CHARMM36m/TIP3P force fields, and the same setups were used as in the pure-MM calculations. All QM/MM dynamic simulations were performed under a canonical (NVT) ensemble at 300 K, and a time step size of 0.5 fs was used.

Despite the presence of two proteins in a given model, only one protein (chain A) was described at the QM/MM level, while the other protein (chain B) remained at the MM level. In general, the QM subsystem consisted of the anions of interest (the carboxylate group of E118 or fluoride) and their immediate surroundings. In particular, water molecules in both the first and second solvation shells of the anions are included. In the simulations, we observed exchanges of water molecules between the solvation shells and bulk solution. Therefore, after every 1 ps, we stopped the simulation, inspected the trajectory, and updated the QM subsystem manually if needed by reclassifying water molecules to be QM or MM. Admittedly, doing so led to discontinuities in the trajectories, as the QM and MM forces were, although often similar, not the same. Fortunately, the reclassifications were typically applied shortly after water molecules left or entered the second solvation shell, while the first solvation shell was always kept at the QM level, mitigating the impact on the anion movements. As such, although this is not an ideal solution, it is a reasonable treatment to keep the computational cost affordable.

2.2.1. E118 Pushing Fext Out of the Pore

With deprotonated E118 in the down position, the MM-equilibrated WT4 model was chosen to simulate the rotation of E118, expelling Fext from the pore. First, the model was further equilibrated at the QM/MM level for 12 ps to relax the localized QM subsystem, which contained the E118 side chain, pE318 backbone, and side chain, Fcen, and initially 10 water molecules surrounding Fcen. Two independent trajectories were simulated using different random seeds, which displayed overall similar equilibrated structures.

Next, one QM/MM-equilibrated structure was employed to explore the path for the rotation of E118 from the down to the up positions. Briefly, the E118 side chain was manually rotated stepwise by changing N–Cα–Cβ–Cγ (χ1), or Cα–Cβ–Cγ–Cδ (χ2), or both as appropriate, such that the carboxylate group moved up each time by ∼0.5 Å in the z direction inside the pore. Between two successive rotational steps, three atoms Cβ, Cγ, and Cδ are restrained to their current positions by harmonic potentials with a force constant of 400 kcal/mol/Å2, and the model was relaxed for 2 ps. During the relaxation, a set of 6 “anchoring” atoms (the Cα atoms of G116, R117, E118, E318, V319, and T320) were also restrained by harmonic potentials with a force constant of 300 kcal/mol/Å2 to prevent the protein from moving along with the E118 side chain. (The anchoring treatment was always applied in the QM/MM simulations in this work unless otherwise noted.) This process, in a sense, treats the rotation of E118 as the translocation of an anion (the deprotonated carboxylate group) through the pore. The E118 rotation was terminated after it reached the up position.

Along the path of E118 rotating up along the pore, the QM subsystem varied accordingly. In general, E118, Fext, and their solvation shells (pore residues and water molecules) were described at the QM level. For example, when Fext was expelled from the external binding site into the extracellular solution, it lost the hydrogen bond with the T320 side chain and instead began interacting with R117; at this point, we incorporated R117 in the extracellular vestibule into the QM subsystem.

2.2.2. Fluoride Reoccupying the External Binding Site

Given the strong interactions between a fluoride ion and pE318, it is reasonable to assume that pE318 helps recruit fluoride ions from the intracellular solution. Therefore, after E118 vacates the central binding site, it should be straightforward for a fluoride ion to diffuse into and repopulate the central binding site. A bigger concern is how the external binding site is reoccupied by fluoride. To address this concern, we decided to simulate a fluoride ion moving from the intracellular entrance of the pore to the external binding site, for which the WT6 model was utilized.

First, the WT6 model was equilibrated at the QM/MM level for 8 ps to relax the QM subsystem, which comprised E118, G317, protonated E318, Y396, Fcen, and initially 6 water molecules. Two trajectories were simulated: one with Fcen restrained at its position by applying harmonic potentials to its Cartesian coordinates and the other without such restraints. The relaxed QM subsystems ended up rather similarly in both trajectories.

Next, with the ending geometry with restraint, we explored the path of the fluoride ion traveling upward along the pore. Specifically, we moved Fcen stepwise to the intracellular pore entrance and then continued moving it upward until it passed the external binding site. For each step, a constant-speed steered molecular dynamic (SMD)53 simulation was performed for 1 ps, where the fluoride ion was pulling upward in the z direction, followed by a 1 ps relaxation of the surroundings with the fluoride ion fixed at its new position. For SMD, the pulling force constant was set to 400 kcal/mol/Å2, and the guiding dummy atom advanced at a speed of 0.5 Å/ps. It should be noted that such a speed is much faster than one would typically use in SMD at the MM level; however, as a compromise between computational accuracy and expense, it might be acceptable here because we are not expecting large-amplitude global conformational changes of the protein during this process.

Along the path of the fluoride ion traveling along the pore, the QM subsystem was updated accordingly. Generally speaking, the fluoride ion and its solvation shells (pore residues and water molecules) were described at the QM level until it expelled E118 out of the external binding site; at this point, we also incorporated R117 and nearby water molecules in the extracellular vestibule into the QM subsystem.

Finally, employing the relaxed structures along the path for the fluoride ion moving up along the pore, we carried out umbrella sampling to assess the associated PMF. The reaction coordinate RC was set to the difference in the z coordinates between the moving fluoride ion and the anchored Cα of E318 (restrained to z = −0.29 Å). The sampling windows are tabulated in Table S2 in the Supporting Information. Note that RC = 0 corresponded to a fluoride ion at the pore entrance instead of at the central binding site. The umbrella potential was applied to the fluoride ion but only in the z direction, i.e., without any constraints in the x and y directions. The force constant of the umbrella potential was chosen to be either 10 or 20 kcal/mol/Å2, depending on the need to achieve a sufficient overlap of the probability distributions between successive sampling windows. Each window was sampled for 7 ps, with the first 1 ps of the trajectory treated as equilibration and discarded. The PMF computed using the WHAM54−56 program with a bin size of 0.1 Å was monitored to ensure a reasonably converged PMF was achieved. We note that overall, the sampling times were very short and that the PMF might not be very accurate, which is unfortunately inevitable due to the high computational costs of QM/MM. Therefore, our goal is limited to qualitative or semiquantitative descriptions of the free-energy landscapes of anion translocation through the pore.

3. Results

3.1. Equilibrated MM Models

The saved trajectories of all MM models were carefully inspected. In all models, the plots of the root-mean-square deviations (RMSDs) of the protein backbone as functions of simulation time quickly plateaued (Figure S1 in the Supporting Information). Overall, the RMSD values are very small (all <2 Å and many ∼1 Å), suggesting that the models were likely equilibrated. The equilibrated proteins also exhibit similar conformations that resembled the experimental structure, as measured by the small (≤2 Å) RMSD values between the saved frames and the crystal structure PDB 6D0J(5) (see Table S2 in the Supporting Information for detailed statistics). The results are in line with the experimental implications that no large-amplitude global conformational change of the protein was involved in ion transports and that local conformational changes may be sufficient to alternate the accesses of the pore to the intra- and extracellular sides.5

It is interesting that energy minimization of WT6 during the model construction moved the deprotonated E118 in the up position into the vacated external binding site (Figure S2 in the Supporting Information), where it stayed stably during the entire equilibration. This can be rationalized by the 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 Fcen 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 Fcen stayed near the central binding site. In contrast, without any restraint, the Fext ions stayed close to their initial positions in the binding sites.

Solvation of fluoride is critical to its binding and transport and is a focus of the present study. Overall, both the intra- and extracellular vestibules became hydrated quickly once the simulations started, bringing water molecules to the anion binding sites. This is exemplified by a snapshot in Figure S4 in the Supporting Information. While the more deeply buried Fext was hydrated by only 2–3 water molecules, Fcen is more extensively hydrated. As illustrations, we show in Figure 2a,b the last frames of Fcen in a protein monomer A of the WT4 and WT6 models, where Fcen interacts directly and indirectly (via a water molecule) with pE318, respectively. In both cases, Fcen was coordinated by 6 hydrogen atoms, 5 of them coming from the water molecules. The last hydrogen atom was provided by the pE318 side chain in WT4 and the Y396 side chain in WT6.

Figure 2.

Figure 2

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Solvation structure of Fcen in protein monomer A. The model is displayed as sticks, except the fluoride ion as a sphere. The protonated E318 is labeled pE318. Distance in Å. Color code: C, cyan; H, white; O, red; N, blue; F, pink. (a) The last frame of WT4 equilibration at the MM level. (b) Similar to panel (a) but for WT6. A water molecule bridged pE318 and Fcen through two hydrogen bonds O1–Hε and Fcen-H1. (c) The last frame of WT4 equilibration at the QM/MM level. (d) Similar to panel (c) but for WT6.

3.2. Fluoride Solvation in QM/MM Simulations

We observed rapid (within 1–2 ps) changes in the solvation structure of Fcen once the QM/MM simulations started from the MM-equilibrated models. In both WT4 and WT6, the trend was the same: a tighter coordination given by the QM descriptions with substantially shorter distances between Fcen and the surrounding hydrogen atoms (Figure 2c,d). In WT4, where Fcen was directly hydrogen-bonded to pE318, the distance Fcen–Hε reduced from 1.61 to 1.46 Å. In WT6, where Fcen was connected to pE318 via a water molecule, both the distances of O1–Hε and Fcen–H1 were shortened by ∼0.2 and 0.3 Å, respectively. The shorter distances led to shrinking coordination numbers of Fcen, which dropped from 6 to 4 in WT4 and from 6 to 5 in WT6. The differences in the solvation structures imply that the employed MM force fields likely underestimate the interactions between a fluoride ion and its surroundings.

To accurately model fluoride ion solvation, it is necessary to include the complete first solvation shell in the QM subsystem. We conducted a number of test simulations with mixed QM and MM water molecules in the first solvation shell. We found that, as the number of QM water molecules dropped, the Fcen–H distance reduces, and proton sharing became increasingly prominent between the fluoride ion and its surroundings, especially when pE318 was in the first solvation shell. One such example of two QM and two MM water molecules is shown in Figure S3 in the Supporting Information, where Hε in pE318 was transferred to Fcen to form a hydrogen fluoride molecule after 100 fs. The trend is easy to understand. When the gas-phase QM calculations (in mechanical embedding QM/MM) were performed with less and less water molecules in the first solvation shell, the fluoride ion was situated in a progressively hydrophobic environment, where the pKa value of hydrogen fluoride continued to increase. On the other hand, when the entire first solvation shell was modeled at the QM level, we hardly observed proton sharing between the fluoride ion and a protonated glutamate.

We suspect that a well-solvated Fcen may be part of the answer to a long-standing puzzle: interactions between fluoride and a protonated glutamate do not cause lockdown in CLCF-eca but do so in CLC-ec1. Although a well-solvated Fcen still interacts strongly with pE318 in CLCF-eca, the interactions are not as strong as those in CLC-ec1 between a fluoride ion and pE148 in the down position inside the pore (Figure 3a). Note that the pore of CLC-ec1 is longer, narrower, and more hydrophobic,11,12 and it does not stay continuously hydrated.57 Thus, the attractions between the fluoride ion and pE148 in CLC-ec1 are much stronger, with proton sharing observed in QM/MM simulations.14,27 As such, we speculate that Fcen in CLCF-eca can be relatively easily pushed into the pore by an incoming fluoride (Figure 3b). Of course, given that the central binding site is located at the top of the intracellular vestibule, being 2–3 Å away from the restricted pore entrance, it is also entirely possible that Fcen and pE318 side chain are pushed aside by a fluoride ion that enters the pore (Figure 3c). At the moment, we do not know which possibility is more likely, and that should be unearthed by further investigations. Nevertheless, either way, the 1:1 fluoride–proton stoichiometry is retained. In contrast, in CLC-ec1, it will be difficult for an incoming chloride ion (which is notably bulkier than fluoride and also interacts much more weakly with a protonated glutamate) to bypass or displace the fluoride trapped inside the pore, and therefore, the transport cycle is disrupted.

Figure 3.

Figure 3

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(a) In the hydrophobic pore of CLC-ec1, a fluoride ion is trapped by strong interactions with protonated E148 (pE148) in the down position, interrupting chloride transport. In CLCF-eca, (b) Fcen in the well-hydrated central binding site interacts less strongly with protonated E318 (pE318) and may be pushed into the pore by another incoming fluoride ion. Alternatively, (c) Fcen and pE318 may be pushed aside to make the pore accessible for an incoming fluoride ion.

3.3. E118 Expelling Fext from the External Binding Site

Sample snapshots extracted from the trajectories of path exploration are shown in Figure 4. Initially, Fext was held in the binding site by the T320 side chain hydroxyl and G116 amide groups. As E118 rotated upward, Fext was pushed out of the binding site, losing contact first with the amide and then with the hydroxyl group. The fluoride ion stayed in the extracellular vestibule while interacting with the R117 side chain, while the external binding site was occupied by the E118 side chain. Continuing the rotation of E118 toward the up position pushed the fluoride further away from the pore. The well-hydrated fluoride ion did not compete with R117 for proton, likely due to the much higher pKa of the protonated R117 side chain. It is conceivable that the fluoride ion would eventually diffuse into the extracellular solution if the simulation time was long enough.

Figure 4.

Figure 4

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Snapshots of the QM subsystem in the path exploration for the E118 upward rotation. Atoms are shown as sticks, except fluoride as a sphere. Color code: C, cyan; H, white; O, red; N, blue; F, pink. The letter “b” in a residue label stands for “backbone only”. The E118 Cδ is marked by a yellow circle, the z coordinate of which is listed for each snapshot. Distances are given in Å.

3.4. Fluoride Repopulates the External Binding Site

Sample snapshots for reloading fluoride into the external binding site are shown in Figure 5, which are extracted from the trajectories of path exploration. Initially, the external binding site was occupied by the E118 side chain, which interacted with the T320 side chain hydroxyl and G116 amide groups. As the fluoride ion moved in, E118 was forced out. When the fluoride ion was in the pore, the V319 amide and two pore water molecules dominated its solvation shell. The solvation shell was gradually changed to the T320 side chain hydroxyl, G116 amide, and water molecules from the external vestibule as the fluoride ion moved into and then went past the external binding site.

Figure 5.

Figure 5

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Snapshots of the QM subsystem in the path exploration for fluoride ion reoccupying the external binding site. All atoms are shown as sticks, except fluoride ion as a sphere. Color code: C, cyan; H, white; O, red; N, blue; F, pink. The letter “b” in a residue label stands for backbone only. The proton that E118 received from R117 is marked by a yellow circle. The reaction coordinate is RC = z(F) – z(E318Cα).

Unexpectedly, E118, which had just been forced out of the external binding site by the incoming fluoride, accepted a proton from R117 to become protonated. This was in sharp contrast with the simulations of E118 expelling Fext, where E118 in the up position did not take a proton from R117. We suspect that the arrival of the negatively charged fluoride in the external binding site upshifted E118′s pKa, facilitating its protonation. This agrees with the early PB electrostatic analysis by Chon et al.14 In contrast, in the simulations of E118 expelling Fext, the well-hydrated fluoride in the external vestibule was significantly screened and thus has a much weaker impact on the protonation of E118. The protonation of E118 naturally starts the next transport cycle.

It may be tempting to think that R117 is necessary for CLCF to function. However, in the absence of R117, a proton from the extracellular solution may still reach E118 through water wires because the extracellular vestibule is wide open to the bulk solution and remained well solvated in our simulations. Therefore, we speculate that R117 may serve as an “antenna” that helps collect proton from the extracellular solution (and temporally stores the proton) for E118 protonation. In other words, although R117 improves the efficiency, its removal should not be fatal.

The PMF of fluoride traveling through the pore is plotted in Figure 6. Overall, the PMF was divided into two sections. The first section extends from RC = 0 to RC ∼ 3.5 Å and sees the fluoride ion displace the E118 side chain in the external binding site. A free-energy barrier of ∼13 kcal/mol was found. Beyond an RC of ∼ 3.5 Å lies the second section, which corresponds to the fluoride ion advancing past the external binding site toward the external vestibule. An additional barrier of ∼7 kcal/mol was recorded before the PMF curves leveled out. However, the second section may not be physiologically relevant because, according to the windmill mechanism, the fluoride ion in the external binding site is expected to be expelled by the rotating E118 side chain in the next transport cycle.

Figure 6.

Figure 6

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PMF of fluoride moving through the pore. The external binding site (Sext) is at an RC of ∼ 3.5 Å. The reaction coordinate is RC = z(F) – z(E318Cα). Two curves were shown based on simulation times of 6 and 7 ps per window, respectively.

4. Discussion and Conclusions

The present contribution enriches our knowledge of the (original) windmill mechanism of CLCF (Figure 7). The earlier calculations by Carloni and co-workers20,21 investigated proton translocation (states a to b) and Fcen-assisted release (states b to c), and here, we study E118 expelling Fext and (states d to e) and the repopulation of the external binding site by fluoride (states e to a). Combining the results from both studies, the following picture of proton-coupled fluoride transport in CLCF-eca emerges: the cycle starts with pE118 in the up position and Fcen and Fext loaded (state a). An almost barrierless clockwise rotation of the pE118 side chain along the proton pathway to the down position brings a proton from the extracellular to intracellular solutions (state b). The arrival of pE118 near the intracellular pore entrance prompts Fcen to receive a proton from pE318 and is subsequently released as a HF molecule into the intracellular solution, leaving pE118 and E318 (state c). Afterward, proton transfer from pE118 to E318, presumably with a very low barrier, gives rise to p318E and E118 (state d). Continuing the clockwise rotation upward along the anion pathway, E118 pushes Fext out of the pore into the extracellular solution, while pE318 recruits Fcen from the intracellular solution (state e). Next, another intracellular fluoride ion comes in, which either moves up to occupy the external binding site directly, bypassing Fcen, or seizes the central binding site from Fcen, forcing the latter to take the external binding sites. The reload of Fext expels E118 from the external binding site and prompts its protonation, making it ready for the next cycle (back to state a). The net outcome of each cycle is the intake of one proton and discharge of one fluoride ion. Unlike the partially overlapped proton and anion pathways in CLC-ec1, here the proton and anion pathways are largely separate, and the alternate accesses of the ion pathways to the intracellular and extracellular solutions are achieved by the E118 side-chain rotation. For the anion translocation, the central theme is the competitions of the external binding site between fluoride and the deprotonated E118.

Figure 7.

Figure 7

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Transport steps in the (original) windmill mechanism,5 with the Fcen-assisted proton release20,21 included. Only E118 and E318 carboxyl groups (as sticks; H, white; C, cyan; O, red) and fluoride groups (pink spheres) are shown. The “p” in the residue label stands for “protonated”.

Furthermore, we now have a better understanding of the mysterious E318 near the central binding site. In particular, there has been a concern that the strong interactions between fluoride and pE318 may cause the fluoride to be trapped in the binding site, leading to a lockdown, as what had been found in CLC-ec1.13,14,21,27 Our QM/MM models suggest that such a lockdown is unlikely in CLCF-eca for two reasons: (i) E318 is located outside the pore, and (ii) the extensive solvation of fluoride prevents it from binding too strongly to pE318. The external binding site can be repopulated by the incoming fluoride pushing Fcen either aside or into the pore, and the transport cycle is not disrupted. Such a picture differs remarkably from what happens in CLC-ec1, where the fluoride ion is trapped by pE148 deep in the largely hydrophobic pore. In short, it is the “location, location, location” that makes a difference.

One may ask, then: What role does a protonated E318 play? We speculate that it helps recruit fluoride from the intracellular solution into the central binding site (after E118 leaves to expel Fext), a step that is necessary for the binding sites to be repopulated. In the absence of E318, fluoride can still be recruited by Y396, although likely less efficiently. Furthermore, the presence of a titratable anion (fluoride) in the central binding site can assist the release of the proton carried into the intracellular vestibule by E118, as demonstrated by Carloni and co-workers.20,21 Of course, in the absence of Fcen, the proton can still be released into the well-solvated intracellular vestibule and eventually into the intracellular solution through water wires, albeit less efficiently. Both rationales are consistent with that mutants E318A and E318Q show reduced proton influx rates and lower fluoride efflux rates.5 However, it is not apparent to us why E318Q, which can help fluoride bind in the central binding site, exhibits transport activities reduced to those of E318A, which cannot. More studies are required to figure this out.

Our simulations confirm that both T320 and Y396 side chains are important in the fluoride solvation in the external and central binding sites, respectively. T320 is more critical because it also interacts favorably with deprotonated E118 in the external binding site. These findings are consistent with the experimental results that deleting either side chain through mutation to alanine impedes ion transports by CLCF-eca, and the impacts are more substantial in T320A than in Y396A.5

The PMF calculations in this work estimated a free-energy barrier of ∼13 kcal/mol for fluoride repopulating the external binding site. Considering the short sampling times in our simulations, to be on the safe side, we would put the barrier in the range of 10–15 kcal/mol. 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. (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.

Acknowledgments

This work was supported by the National Institute of General Medical Sciences (1R15GM141728-01). This work used the Expanse at San Diego Supercomputer Center through allocations BIO230001 and CHE230017 from the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program, which is supported by the National Science Foundation grants #2138259, #2138286, #2138307, #2137603, and #2138296. The authors thank Adam Duster, Sam Fredrick, Sierra Knodle, Jennifer Nguyen, and Natalie Schultz for helpful discussion.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.4c00079.

  • Additional details about the QM/MM calculations; list of equilibrated MM models; protein backbone RMSD (Å) between simulated and experimental structures; list of umbrella sampling windows; protein backbone RMSD (Å) vs MM equilibration time; E118 conformation changes during the minimization of model WT6; fluoride RMSD (Å) vs simulation time in WT1 equilibration; sample snapshot of hydration of the anion binding sites in MM equilibrations of model WT4; and proton sharing in QM/MM due to limited solvation of fluoride ion (PDF)

The authors declare no competing financial interest.

Supplementary Material

jp4c00079_si_001.pdf (1.7MB, pdf)

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