Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Simulation of pH-Dependent Conformational Transitions in Membrane Proteins: The CLC-ec1 Cl^-/H⁺ Antiporter.

ABSTRACT: 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 Glu_in and Glu_ex.

SUBMITTER: Kots E

PROVIDER: S-EPMC8625536 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The coupled proton transport in the ClC-ec1 Cl(-)/H(+) antiporter.

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

Multiscale Kinetic Modeling Reveals an Ensemble of Cl-/H+ Exchange Pathways in ClC-ec1 Antiporter.

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

The role of conformational change and key glutamic acid residues in the ClC-ec1 antiporter.

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

The ClC-3 Cl-/H+ antiporter becomes uncoupled at low extracellular pH.

Project description:Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH <or= 5.5. ICl(acid) was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl(acid) was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E(rev)) of I(ClC-3) between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl(-) and H(+) transport become uncoupled at low pH. At pH 4.0 E(rev) was completely Cl(-) dependent (55.8 +/- 2.3 mV/decade). Several findings linked ClC-3 with native ICl(acid); 1) RNA interference directed at ClC-3 message reduced native ICl(acid); 2) removal of the extracellular "fast gate" (E224A) produced large currents that were pH-insensitive; and 3) wild-type I(ClC-3) and ICl(acid) were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10-500 microm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I(ClC-3) and from native ICl(acid) by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl(acid). Low pH uncouples Cl(-)/H(+) transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl(-)/H(+) antiporters and perhaps for pH regulation in highly acidic intracellular compartments.

| S-EPMC2807314 | biostudies-literature

A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl-/H+ transport cycle.

Project description:Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl- transport that reconciles existing data on all CLC-type proteins.

| S-EPMC7253180 | biostudies-literature

13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.

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

Proton transport pathway in the ClC Cl-/H+ antiporter.

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

Antiport mechanism for Cl(-)/H(+) in ClC-ec1 from normal-mode analysis.

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

Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR.

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

Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter.

Project description:Multiscale reactive molecular dynamics simulations are used to study proton transport through the central region of ClC-ec1, a widely studied ClC transporter that enables the stoichiometric exchange of 2 Cl(-) ions for 1 proton (H(+)). It has long been known that both Cl(-) and proton transport occur through partially congruent pathways, and that their exchange is strictly coupled. However, the nature of this coupling and the mechanism of antiporting remain topics of debate. Here multiscale simulations have been used to characterize proton transport between E203 (Glu(in)) and E148 (Glu(ex)), the internal and external intermediate proton binding sites, respectively. Free energy profiles are presented, explicitly accounting for the binding of Cl(-) along the central pathway, the dynamically coupled hydration changes of the central region, and conformational changes of Glu(in) and Glu(ex). We find that proton transport between Glu(in) and Glu(ex) is possible in both the presence and absence of Cl(-) in the central binding site, although it is facilitated by the anion presence. These results support the notion that the requisite coupling between Cl(-) and proton transport occurs elsewhere (e.g., during proton uptake or release). In addition, proton transport is explored in the E203K mutant, which maintains proton permeation despite the substitution of a basic residue for Glu(in). This collection of calculations provides for the first time, to our knowledge, a detailed picture of the proton transport mechanism in the central region of ClC-ec1 at a molecular level.

| S-EPMC4816718 | biostudies-literature