The Coupled Proton Transport in the ClC-ec1 Cl⁻/H⁺ Antiporter

Abstract

Using a reactive molecular dynamics simulation methodology, the free energy barrier for water-mediated proton transport between the two proton gating residues Glu²⁰³ and Glu¹⁴⁸ in the ClC-ec1 antiporter, including the Grotthuss mechanism of proton hopping, was calculated. Three different chloride-binding states, with 1), both the central and internal Cl⁻, 2), the central Cl⁻ only, and 3), the internal Cl⁻ only, were considered and the coupling to the H⁺ transport studied. The results show that both the central and internal Cl⁻ are essential for the proton transport from Glu²⁰³ to Glu¹⁴⁸ to have a favorite free energy driving force. The rotation of the Glu¹⁴⁸ side chain was also found to be independent of the internal chloride binding state. These results emphasize the importance of the 2:1 stoichiometry of this well-studied Cl⁻/H⁺ antiporter.

The ClC group of proteins includes both Cl⁻ channels and Cl⁻/ H⁺ antiporters (1). For the channels, Cl⁻ diffuses down the electrochemical gradient passively, whereas in antiporters, Cl⁻ is transported against the electrochemical gradient by coupling to the downhill movement of H⁺, or vice versa. The ClC-ec1 protein found in Escherichia coli is one of the antiporters in this family that stoichiometrically exchanges two chlorides with one proton (2). A high-resolution crystal structure of ClC-ec1 was determined by Dutzler et al. (3) that stimulated several studies on this protein using various methods (4, 5, 6, 7, 8, 9).

For the ClC-ec1 antiporter, it has been found that the proton entering and leaving the protein channel or crevice is gated by two residues: Glu²⁰³ on the internal side (10, 11) and Glu¹⁴⁸ on the external side (7, 12), respectively. In the crystal structure of wild-type ClC-ec1, the Glu²⁰³ side-chain χ2 angle has a value of ∼60°, which was found to be favored for the deprotonated Glu²⁰³ (11). Once a proton binds to the Glu²⁰³ side-chain carboxyl group, the side chain can reorient to a configuration where the χ2 angle has a value of ∼180°. This reorientation brings the proton into the protein crevice (11). The Glu¹⁴⁸ on the other side of the membrane is believed to behave similarly. The closed state with an χ2 angle of 60° was preferred when it was deprotonated. Once Glu¹⁴⁸ is protonated, its open state (with χ2 angle of 180°) is accessible (7, 12). The rotation of the Glu¹⁴⁸ side chain carries the proton from the protein crevice to the external side of the cell membrane, followed by the delivery of the proton to the bulk solution. Between the two gating glutamate residues, the proton may be conducted along a metastable water chain (11) (no static water molecules are observed in the ClC-ec1 crystal structure).

The conductance of Cl⁻ ions through the ClC channels or antiporters has also been widely studied (13, 14, 15). Three Cl⁻ binding sites have been identified for ClC-ec1 and were generally labeled as external, central, and internal sites, respectively (6, 12, 16). In the crystal structure, the external site was occupied by the deprotonated Glu¹⁴⁸ side chain, whereas Cl⁻ ions were observed at the central and internal sites. Chloride transport is believed to follow a separate pathway from that of proton transport, except the location at Glu¹⁴⁸, where the two pathways meet (10).

Experimental studies on ClC-ec1 mutants have provided a picture of the stoichiometrically coupled mechanism (17, 18, 19). Recently, a Cl⁻/H⁺ exchange cycle was proposed by Miller and Nguitragool (20). The central Cl⁻ plays a key role in the proton transport from Glu²⁰³ to Glu¹⁴⁸ in this mechanism. Based on normal mode analysis, Miloshevsky et al. (8) have suggested that the proton transport is assisted by both the internal and the central Cl⁻ ions.

To clarify the mechanism of the coupled Cl⁻/H⁺ transport in the ClC-ec1 antiporter, the proton transport between Glu²⁰³ and Glu¹⁴⁸ was studied in the current work using molecular dynamics simulations for different chloride binding states: with both central and internal binding sites occupied, only the central binding site occupied, and only the internal binding site occupied (see Fig. 1). The proton transport was explicitly treated using the second-generation multistate empirical valence bond model (21) (see the Supporting Material for a description of methods). The free energy profiles for proton transport are shown in Fig. 2 (see the Supporting Material for details).

Figure 1.

Local structure of the ClC-ec1 Cl⁻/H+ antiporter. The waters colored lightly share an excess proton in this figure. (Black arrow) Definition of the reaction coordinate (RC) used in the potential of mean force calculation. A detailed picture and RC definition can be found in the Supporting Material.

Figure 2.

Calculated potential of mean force (PMF) for the excess proton transport along the water chain reaction coordinate (RC) from the protonated Glu²⁰³ (beginning) to protonated Glu¹⁴⁸ (end) states for each Cl⁻ binding state.

When only the internal Cl⁻ was included in the protein, the potential of mean force (PMF) profile is higher than that of the central Cl⁻-only curve. As shown in Fig. 2, differences as large as 4 kcal/mol are observed when the proton is close to Glu¹⁴⁸. This result clearly shows the important role of the central Cl⁻ in helping drive the proton flux to Glu¹⁴⁸ from Glu²⁰³ through the water chain (11, 16, 19, 20).

Importantly, when both the internal and central Cl⁻ ions are in their binding sites, the proton transport PMF profile is below that of the central Cl⁻-only case everywhere, especially for the first half (RC < 4 Å). It can be seen from Fig. 2 that the internal Cl⁻ lowers the deprotonation barrier from Glu²⁰³ by 3–4 kcal/mol. For the region with RC = 4–6 Å, the central Cl⁻-only curve is very close to that of both the central and internal Cl⁻ curve, indicating that the central Cl⁻ dominates the electrostatic interaction in this region. Interestingly, relative to the central Cl⁻ case, the additional internal Cl⁻ lowers the free energy of protonated Glu¹⁴⁸ state by 4.9 kcal/mol. As a result, the proton transport from Glu²⁰³ to Glu¹⁴⁸ is found to be thermodynamically favored (exothermic) when both the central and the internal Cl⁻ ions are in their respective binding sites. With only one Cl⁻ bound in the protein, either the central one or the internal one, the protonated Glu¹⁴⁸ state is significantly higher in free energy. This indicates that in addition to the central Cl⁻, the internal Cl⁻ plays an essential role in the proton transport, thus emphasizing the importance of the 2:1 Cl⁻/H⁺ stoichiometry.

Recently, Feng et al. (22) have identified a crystal structure of a different antiporter in the ClC family, ClC-cm, which transports Cl⁻ and H⁺ stoichiometrically similarly to ClC-ec1. As for ClC-ec1, three Cl⁻ binding sites were observed in ClC-cm and a similar gating mechanism was suggested. However, the external gating glutamate occupies the chloride central binding site when it is deprotonated, instead of the external one in ClC-ec1. In this case, the internal Cl⁻ may play an important role in assisting the proton translocation between the gating residues.

It should be noted that the role the central and internal chlorides play in assisting the proton transport is not simply additive. One example of such an effect is the protein residue configurational change upon different Cl⁻ binding states. As demonstrated in Fig. S3 of the Supporting Material, when the central binding site is occupied by a Cl⁻ ion, it is coordinated by the hydroxyl group of the Y445 side chain. This way, the Cl⁻ ion and Y445 side chain are stabilized by each other. However, when the central binding site is empty and the internal Cl⁻ binding site is occupied, the side chain of Y445 was found to flip and coordinate with the internal Cl⁻. Such a configurational change modifies the local environment and may affect the proton transport (6, 16).

At the end of the proton transport pathway, the proton binds to Glu¹⁴⁸, which regulates the proton transport to the external solution. It has been suggested that the closed state is preferred for Glu¹⁴⁸ when it is deprotonated, whereas the open state is accessible when it is protonated (7, 12). Using classical molecular dynamics, the PMFs for the E148 side-chain rotation were calculated for both the protonated and deprotonated states under different chloride binding states. The results are shown in Fig. S4. For the deprotonated Glu¹⁴⁸, a minimum is observed at χ2 = ∼60° and the open state is forbidden, whereas for the protonated Glu¹⁴⁸, the open state is accessible with a rotation barrier of 4 kcal/mol. The PMFs calculated with one (central) or two (central and internal) chloride ions in the simulation systems show similar results.

In summary, using reactive molecular dynamics simulations, the free energy profile (PMF) associated with the proton transport between the two proton gating residues, Glu²⁰³ to Glu¹⁴⁸, in the ClC-ec1 antiporter was calculated. The Grotthuss mechanism of proton hopping was explicitly included using the reactive empirical valence bond model. Three different chloride binding states, with 1), both the central Cl⁻ and the internal Cl⁻, 2), the central Cl⁻ only, and 3), the internal Cl⁻ only, were considered, and the coupled proton transport was studied.

The calculated results demonstrate that the central chloride ion plays an essential role in assisting the proton transport between the two glutamate residues, especially when the proton is close to Glu¹⁴⁸ (16, 19, 20). In addition, the results show that the chloride ion occupying the internal binding site, although relatively far from the proton transport path, provides an essential contribution to a favorable free energy for the proton transport process. When both the internal and the central binding sites are occupied by Cl⁻ ions, the proton transport from Glu²⁰³ to Glu¹⁴⁸ experiences a substantial driving force, thus emphasizing the importance of the 2:1 Cl⁻/H⁺ stoichiometry.

The PMFs associated with Glu¹⁴⁸ side-chain rotation were also calculated with one or two chlorides in their binding sites. It was found that the open state is forbidden for the deprotonated state whereas it is accessible for the protonated state, consistent with previous suggestions (7, 12). The barrier from the closed state to the open state was found to be ∼4 kcal/mol, which is independent of the internal chloride binding state.

Editor: Gerhard Hummer.