A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl-/H+ transport cycle.
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ABSTRACT:
SUBMITTER: Chavan TS
PROVIDER: S-EPMC7253180 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Using a reactive molecular dynamics simulation methodology, the free energy barrier for water-mediated proton transport between the two proton gating residues Glu(203) and Glu(148) 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 Glu(203) to Glu(148) to have a favorite free energy driving force. The rotation of the Glu(148) 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.
| S-EPMC3218320 | biostudies-literature
Project description:Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl- anion and a proton (H+), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme-termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Gluin and Gluex.
| S-EPMC8625536 | biostudies-literature
Project description:CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).
| S-EPMC4398623 | biostudies-literature
Project description:The triple glutamine (Q) mutant (QQQ) structure of a Cl-/H+ antiporter from Escherichia coli (ClC-ec1) displaying a novel backbone arrangement has been used to challenge the long-held notion that Cl-/H+ antiporters do not operate through large conformational motions. The QQQ mutant substitutes the glutamine residue for an external glutamate E148, an internal glutamate E203, and a third glutamate E113 that hydrogen-bonds with E203. However, it is unknown if QQQ represents a physiologically relevant state, as well as how the protonation of the wild-type glutamates relates to the global dynamics. We herein apply continuous constant-pH molecular dynamics to investigate the H+-coupled dynamics of ClC-ec1. Although any large-scale conformational rearrangement upon acidification would be due to the accumulation of excess charge within the protein, protonation of the glutamates significantly impacts mainly the local structure and dynamics. Despite the fact that the extracellular pore enlarges at acidic pHs, an occluded ClC-ec1 within the active pH range of 3.5-7.5 requires a protonated E148 to facilitate extracellular Cl- release. E203 is also involved in the intracellular H+ transfer as an H+ acceptor. The water wire connection of E148 with the intracellular solution is regulated by the charge states of the E113/E203 dyad with coupled proton titration. However, the dynamics extracted from our simulations are not QQQ-like, indicating that the QQQ mutant does not represent the behavior of the wild-type ClC-ec1. These findings reinforce the necessity of having a protonatable residue at the E203 position in ClC-ec1 and suggest that a higher level of complexity exists for the intracellular H+ transfer in Cl-/H+ antiporters.
| S-EPMC10111279 | biostudies-literature
Project description:ClC chloride channels and transporters play major roles in cellular excitability, epithelial salt transport, volume, pH, and blood pressure regulation. One family member, ClC-ec1 from Escherichia coli, has been structurally resolved crystallographically and subjected to intensive mutagenetic, crystallographic, and electrophysiological studies. It functions as a Cl(-)/H(+) antiporter, not a Cl(-) channel; however, the molecular mechanism for Cl(-)/H(+) exchange is largely unknown. Using all-atom normal-mode analysis to explore possible mechanisms for this antiport, we propose that Cl(-)/H(+) exchange involves a conformational cycle of alternating exposure of Cl(-) and H(+) binding sites of both ClC pores to the two sides of the membrane. Both pores switch simultaneously from facing outward to facing inward, reminiscent of the standard alternating-access mechanism, which may have direct implications for eukaryotic Cl(-)/H(+) transporters and Cl(-) channels.
| S-EPMC2849085 | biostudies-literature
Project description:Cl(-)/H(+) antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl(-), Br(-), I(-), NO3(-) and SCN(-), but they seem to be very selective against F(-). The recent discovery of a new CLC clade of F(-)/H(+) antiporters, which are highly selective for F(-) over Cl(-), led us to investigate the mechanism of Cl(-)-over-F(-) selectivity by a CLC Cl(-)/H(+) antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography, we show that F(-) binds in the Cl(-) transport pathway with affinity similar to Cl(-) but stalls the transport cycle. Examination of various mutant antiporters implies a 'lock-down' mechanism of F(-) inhibition, in which F(-), by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.
| S-EPMC3805709 | biostudies-literature
Project description:The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl(-) permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent (19)F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl(-) permeation pathway and show the usefulness of (19)F NMR for studying conformational changes in membrane proteins of known structure.
| S-EPMC2771095 | biostudies-literature
Project description:Despite several years of research, the ion exchange mechanisms in chloride/proton antiporters and many other coupled transporters are not yet understood at the molecular level. Here, we present a novel approach to kinetic modeling and apply it to ion exchange in ClC-ec1. Our multiscale kinetic model is developed by (1) calculating the state-to-state rate coefficients with reactive and polarizable molecular dynamics simulations, (2) optimizing these rates in a global kinetic network, and (3) predicting new electrophysiological results. The model shows that the robust Cl:H exchange ratio (2.2:1) can indeed arise from kinetic coupling without large protein conformational changes, indicating a possible facile evolutionary connection to chloride channels. The E148 amino acid residue is shown to couple chloride and proton transport through protonation-dependent blockage of the central anion binding site and an anion-dependent pKa value, which influences proton transport. The results demonstrate how an ensemble of different exchange pathways, as opposed to a single series of transitions, culminates in the macroscopic observables of the antiporter, such as transport rates, chloride/proton stoichiometry, and pH dependence.
| S-EPMC5812667 | biostudies-literature
Project description:Cl–/H+ transporters of the CLC superfamily form a ubiquitous class of membrane proteins that catalyze stoichiometrically coupled exchange of Cl– and H+ across biological membranes. CLC transporters exchange H+ for halides and certain polyatomic anions, but exclude cations, F–, and larger physiological anions, such as PO43– and SO42–. Despite comparable transport rates of different anions, the H+ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter revealed that Cl– binding to the central anion-binding site (Scen) is crucial for the anion-coupled H+ transport. Here, we show that Cl–, F–, NO3–, and SCN– display distinct binding coordinations at the Scen site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H+ transfer sites (Gluin and Gluex), in the presence of different anions. While continuous water wires are frequently observed in the presence of physiologically transported Cl–, binding of F– or NO3– leads to the formation of pseudo-water-wires that are substantially different from the wires formed with Cl–. Binding of SCN–, however, eliminates the water wires altogether. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H+ transport to the transport of anions other than Cl–.
| S-EPMC4898471 | biostudies-literature
Project description:A fundamental question concerning the ClC Cl-/H+ antiporters is the nature of their proton transport (PT) pathway. We addressed this issue by using a novel computational methodology capable of describing the explicit PT dynamics in the ClC-ec1 protein. The main result is that the Glu203 residue delivers a proton from the intracellular solution to the core of ClC-ec1 via a rotation of its side chain and subsequent acid dissociation. After reorientation of the Glu203 side chain, a transient water-mediated PT pathway between Glu203 and Glu148 is established that is able to receive and translocate the proton via Grotthuss shuttling after deprotonation of Glu203. A molecular-dynamics simulation of an explicit hydrated excess proton in this pathway suggests that a negatively charged Glu148 and the central Cl- ion act together to drive H+ to the extracellular side of the membrane. This finding is consistent with the experimental result that Cl- binding to the central site facilitates the proton movement. A calculation of the PT free-energy barrier for the ClC-ec1 E203V mutant also supports the proposal that a dissociable residue is required at this position for efficient delivery of H+ to the protein interior, in agreement with recent experimental results.
| S-EPMC2711357 | biostudies-literature