Probing the conformation of a conserved glutamic acid within the Cl− pathway of a CLC H+/Cl− exchanger

Vien, Malvin; Basilio, Daniel; Leisle, Lilia; Accardi, Alessio

doi:10.1085/jgp.201611682

Cited by 19 publications

(27 citation statements)

References 24 publications

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“…1C) and reflect the reduced ability of the shorter aspartate side chain to reach the central anion binding site (S cen ). Recently, it was also suggested that CLC-ec1 may adopt a cmCLC-like state by the high external Cl − concentrationinduced conformational locking of the Glu ex at the S cen (25). A computational study also proposed a transport cycle in which the Glu ex could occupy the S cen in CLC-ec1 (26).…”

Section: Significancementioning

confidence: 99%

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl ⁻ /H ⁺ antiporter

Park

Lee

Lim

2019

Proc. Natl. Acad. Sci. U.S.A.

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The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl− channels and Cl−/H+ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Gluex, could explain the transport cycle of CLC-type Cl−/H+ antiporters. However, the presence of multiple conformations (Up, Middle, and Down) of the Gluex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae. Thus, we aimed to investigate further the heterogeneity of Gluex-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Gluex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the “Midlow” conformation. We also found that an extra anion can be located above the external Cl−-binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl−-binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Gluex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.

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Section: Significancementioning

confidence: 99%

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl ⁻ /H ⁺ antiporter

Park

Lee

Lim

2019

Proc. Natl. Acad. Sci. U.S.A.

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“…These results suggest that Sxet is a genuine low-affinity anion binding site in CLC-ec1, which can be occupied by either a water molecule or halide ion depending on the anion concentration. Thus, it is possible to imagine that the conformational change of Gluex from Middle to Up-conformations might be hampered by extra Clbinding at Sxet, which induces the slowed ion transport in high [Cl -] condition as observed previously 25 .…”

Section: The Presence Of Extra Anion Binding Site Above the External mentioning

confidence: 85%

“…A recent study showed that high external Clconcentration (above 300 mM) significantly slowed both Cland H + transports in CLC-ec1 by limiting conformational changes of the Gluex from Down-to Up-conformations 25 . Also, a computational study on CLC-ec1 suggested an extra Cl-binding site above the Sext 36 .…”

Section: The Presence Of Extra Anion Binding Site Above the External mentioning

confidence: 99%

Structural capture of an intermediate transport state of a CLC CI^-/H⁺ antiporter

Park

Lim

2018

Preprint

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The CLC family proteins are involved in a variety of cellular processes, where chloride homeostasis needs to be controlled. Two distinct classes of CLC proteins, Clchannels and Cl -/H + antiporters, have been functionally and structurally investigated over the last several decades. Recent studies have revealed that the conformational heterogeneity of the critical glutamate residue, Gluex could explain the transport cycle of CLC-type Cl -/H + antiporters. However, the presence of multiple conformations of the Gluex has been suggested from combined structural snapshots of two different CLC antiporters. Thus, we aimed to investigate the presence of these three intermediate conformations in CLC-ec1, the most deeply studied CLC at both functional and structural levels. By comparing crystal structures of E148D, E148A mutant and wildtype CLC-ec1 with varying anion concentrations, we suggest that the Gluex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.Recent studies on the mutation of the external glutamate (Gluex) in CLC-type Cl -/H + antiporters to aspartate, an amino acid with a methylene (-CH2 group) shorter and a similar pKa value, reported extremely slow ion transport in both CLC-ec1 and cmCLC 19 . Moreover, a mutation at the corresponding glutamate (E166D) in the CLC-0 channel reduced singlechannel conductance as well as drastically decreased open probability 23,24 . These previous observations support the idea that the Gluex has three conformational states (Up-, Middle-, and Down-conformations; Fig. 1C), and reflect the penalty of the shorter aspartate side chain to reach the Scen. Recently, it was also suggested that CLC-ec1 could adopt a cmCLC-like state by a conformational locking of the Gluex at the central anion binding site (Scen), which is induced by high external Clconcentration 25 . Computational study also proposed a transport cycle in which the Gluex could occupy the Scen in CLC-ec1 26 .However, the presence of cmCLC-like "Down" conformation, the Gluex occupying the Scen, has not been observed in CLC-ec1 structure, only in cmCLC. Thus, we aimed to tackle the following: (1) elucidating the structural change in the E148D mutant CLC-ec1, and explain the mechanistical consequences of E148D mutation, if it exists, (2) visualizing the structural transition occurring due to Asp148 protonation of the E148D mutant by comparing its structure with E148N mutant, and (3) obtaining structural insight into the conformational changes of the Gluex, and especially into its presence at the Scen in CLC-ec1 during the transport cycle.Structural and functional examinations of E148D and E148N CLC-ec1 revealed unexpected rotameric change upon protonation/deprotonation of Asp148, which resulted in limited solution accessibility to the Asp148 residue and could slow ion transport. From the comparison of the E148D mutant and halide-free wild-type structures, we provide evidence for the presence of a new intermediate "Mid-low" state in the transition between "Middle" and "Down" confo...

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“…The CLC transporter structures represent a closed conformational state in which bound chloride (Cl − ) is occluded by proteinaceous "gates" that block exit to either the outer (extracellular) or inner (intracellular) side of the protein. The outer gate is formed in part by a conserved glutamate residue, GLU ex [19][20][21][22][23][24][25][26] (Fig 1). When mutated to GLN (to resemble the protonated GLU), this side chain rotates outwards and partially unblocks the Cl − permeation pathway [19].…”

Section: Introductionmentioning

confidence: 99%

Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating

et al. 2020

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This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLU ex. Both additional states involve conformational changes that orient GLU ex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLU ex results in an open, or near-open, channel pore. The equilibrium population of this state is low (*1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLU ex rotates to occlude the channel pore. This state, which has a low equilibrium population (*1%), is only accessible when GLU ex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLU ex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.

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Probing the conformation of a conserved glutamic acid within the Cl− pathway of a CLC H+/Cl− exchanger

Cited by 19 publications

References 24 publications

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl ⁻ /H ⁺ antiporter

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl ⁻ /H ⁺ antiporter

Structural capture of an intermediate transport state of a CLC CI^-/H⁺ antiporter

Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating

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Probing the conformation of a conserved glutamic acid within the Cl− pathway of a CLC H+/Cl− exchanger

Cited by 19 publications

References 24 publications

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl − /H + antiporter

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl − /H + antiporter

Structural capture of an intermediate transport state of a CLC CI-/H+ antiporter

Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating

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Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl ⁻ /H ⁺ antiporter

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl ⁻ /H ⁺ antiporter

Structural capture of an intermediate transport state of a CLC CI^-/H⁺ antiporter