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

Abstract

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.

Single-molecule FRET monitors CLC transporter conformation and subunit independence

“…In addition, the structure involves a rearrangement of the three glutamine residues: Gluex (Glnex), the H + -transfer residue, reaches inwards for H + exchange to the intracellular side, while the intracellular Gln residues separate from one another, allowing for a water/H + pathway from the intracellular side ( Figure 1B). The relevance of this conformation to the wild-type (WT) protein was confirmed using double electron-electron resonance (DEER) spectroscopy experiments to compare conformations of WT and QQQ mutant proteins; these experiments revealed that wild-type CLC-ec1 adopts a QQQ-like conformation when the pH is lowered from 7.5 to 4.5 to protonate the key glutamate residues (Chavan et al, 2020).…”

“…Mutagenesis experiments to generate the E377C/H234C, Y75C/H234C, and Y75C/E377C mutants were carried out in a cysteine-less background (C85A/C302A/C347S) (Nguitragool and Miller, 2007), in either an otherwise WT CLC-ec1 background or in a "QQQ" background (E148Q/E203Q/E113Q) (Chavan et al, 2020). Mutants were verified by sequencing the entire coding region of the gene.…”

“…Gluex acts as both a "gate" to the Cl --permeation pathway and as a H + -transfer residue. The passage of Cland H + occurs along pathways that are shared at the extracellular portion of the protein and then diverge towards the intracellular side (Accardi et al, 2005;Accardi, 2015;Chavan et al, 2020). In the channel homologs, H + transfer is uncoupled from Clpermeation, such that thousands of Clions are transported for every H + , and thus H + transport can be measured only indirectly Maduke, 2008, 2009).…”

“…Most of these show the same overall conformation -an "occluded" state in which the bound Clsubstrate is blocked from both the extracellular and intracellular solutions. Recently, a crystal structure of a CLC-ec1 triple mutant ("QQQ"to mimic protonation of three key glutamate residues) revealed the first molecular details of the CLC-ec1 outward-facing conformational state, with the permeation pathway open for Clto exchange to the extracellular side (Chovancova et al, 2012;Chavan et al, 2020) ( Figure 1B). In addition, the structure involves a rearrangement of the three glutamine residues: Gluex (Glnex), the H + -transfer residue, reaches inwards for H + exchange to the intracellular side, while the intracellular Gln residues separate from one another, allowing for a water/H + pathway from the intracellular side ( Figure 1B).…”

“…In addition, the single-molecule approach confers flexibility in labeling strategies, allowing labeling at multiple sites without concerns of sample heterogeneity, which can thwart ensemble-spectroscopic measurements. For example, the DEER measurements in our previous study used a labeling strategy with one label per subunit, measuring inter-subunit distance distributions (Chavan et al, 2020); this strategy is valuable because it avoids spectral overlap of intra-and inter-subunit distance distributions that would occur with multiple labels per subunit, but it does not provide information about independent movements within a subunit. Here, we design smFRET experiments to monitor conformational change within one subunit of the CLC-ec1 dimer, without interference from labels on the second subunit.…”