Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter

Khantwal, Chandra M.; Abraham, Sherwin J.; Han, Wei; Jiang, Tao; Chavan, Tanmay; Cheng, Ricky C.; Elvington, Shelley; Liu, Corey W.; Mathews, I.I.; Stein, Richard A.; Mchaourab, Hassane S.; Tajkhorshid, Emad; Maduke, Merritt

doi:10.7554/elife.11189

Cited by 44 publications

(114 citation statements)

References 114 publications

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“…Such an uncoupling mechanism has precedent in ClC-ec1 Glu ex mutations, where uncoupled Cl − transport involves protein conformational change similar to that observed in the coupled transporter. 69 The idea that uncoupled Cl − and SCN − transport share some similarities is consistent with the observation that mutations at Glu in dramatically slow both Cl − and SCN − transport in ClC-5. 14 We note also that our observation of SCN − binding to S cen is not inconsistent with the crystallo-graphic studies, in which the lack of anion density at S cen could reflect high disorder rather than lack of occupancy.…”

Section: Discussionsupporting

confidence: 74%

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl^–/H⁺ Exchanger ClC-ec1

et al. 2016

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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−.

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Section: Discussionsupporting

confidence: 74%

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl^–/H⁺ Exchanger ClC-ec1

et al. 2016

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“…At Helix P, our previous data provide 6 further support for the relevance of the conformational changes observed in QQQ. First, we showed 7 that CLC-ec1 activity is inhibited by cross-linking of D417C, on Helix P, across the dimer interface 8 (Khantwal et al, 2016). Thus, residues D417 must move apart from one another during the transport 9 cycle.…”

Section: Validation Of Qqq Conformational Change In Wt Clc-ec1 Using mentioning

confidence: 95%

“…The extracellular bottleneck to anion permeation is formed in part by Helix N, which together 23 with Helix F forms the anion-selectivity filter (Dutzler et al, 2002). Previously, we proposed that 24 generation of the outward-facing open state involves movement of Helix N in conjunction with its 25 neighbor Helix P (at the dimer interface) to widen this bottleneck (Khantwal et al, 2016). Structural 26 alignment of the QQQ mutant with either E148Q (Figure 3A-C) or WT (Figure 3C) confirms the 27 movement of these helices.…”

Section: Structural Changes Underlying the Opening Of The Extracellulmentioning

confidence: 99%

“…Using a combination of biochemical crosslinking, double electron-electron resonance (DEER) 7 spectroscopy, functional assays, and molecular dynamics (MD) simulations, we concluded that this 8 H + -induced conformational state represents an "outward-facing open" state, an intermediate in the 9 transport cycle that facilitates anion transport to and from the extracellular side (Khantwal et al, 2016). 10…”

Section: Introductionmentioning

confidence: 99%

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Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl⁻/H⁺ transport cycle

Chavan

Cheng

Jiang

et al. 2019

Preprint

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ABSTRACTAmong 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 many experimental findings, there have remained gaps and inconsistencies in our understanding. 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 a state that has improved accessibility for the Cl− substrate from the extracellular side and new conformations for two key glutamate residues. Based on this new structure, together with DEER measurements, MD simulations, and functional studies, we propose a unified model of the CLC transport mechanism that reconciles existing data on all CLC-type proteins.

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“…Here in particular a study unraveling the protonation-dependent conformational dynamics has contributed significantly to the understanding of the underlying transport mechanism [147]. A combined effort of NMR, EPR and MD simulations shed light on global structural changes in the Cl − /H + exchanger family CLC, which are necessary to form the previously unknown outward-facing open conformation [148]. Two studies [149,150] investigated the conformational dynamics of the aspartate/Na + symporter Glt Ph , which is a member of the excitatory amino acid transporter family (EAAT), also referred to as SLC1.…”

Section: Secondary Active Proteinsmentioning

confidence: 99%

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

Wunnicke

Hänelt

2017

Crystals

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Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge.

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Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter

Cited by 44 publications

References 114 publications

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl^–/H⁺ Exchanger ClC-ec1

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl^–/H⁺ Exchanger ClC-ec1

Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl⁻/H⁺ transport cycle

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

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Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter

Cited by 44 publications

References 114 publications

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl–/H+ Exchanger ClC-ec1

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl–/H+ Exchanger ClC-ec1

Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl−/H+ transport cycle

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

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Product

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Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl^–/H⁺ Exchanger ClC-ec1

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl^–/H⁺ Exchanger ClC-ec1

Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl⁻/H⁺ transport cycle