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


The CLC proteins form a broad family of anion-selective transport proteins that includes both channels and exchangers. Despite extensive structural, functional, and computational studies, the transport mechanism of the CLC exchangers remains poorly understood. Several transport models have been proposed but have failed to capture all the key features of these transporters. Multiple CLC crystal structures have suggested that a conserved glutamic acid, Gluex, can adopt three conformations and that the interconversion of its side chain between these states underlies H+/Cl- exchange. One of these states, in which Gluex occupies the central binding site (Scen) while Cl- ions fill the internal and external sites (Sint and Sext), has only been observed in one homologue, the eukaryotic cmCLC. The existence of such a state in other CLCs has not been demonstrated. In this study, we find that during transport, the prototypical prokaryotic CLC exchanger, CLC-ec1, adopts a conformation with functional characteristics that match those predicted for a cmCLC-like state, with Gluex trapped in Scen between two Cl- ions. Transport by CLC-ec1 is reduced when [Cl-] is symmetrically increased on both sides of the membrane and mutations that disrupt the hydrogen bonds stabilizing Gluex in Scen destabilize this trapped state. Furthermore, inhibition of transport by high [Cl-] is abolished in the E148A mutant, in which the Gluex side chain is removed. Collectively, our results suggest that, during the CLC transport cycle, Gluex can occupy Scen as well as the Sext position in which it has been captured crystallographically and that hydrogen bonds with the side chains of residues that coordinate ion binding to Scen play a role in determining the equilibrium between these two conformations.

DOI: 10.1085/jgp.201611682

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@inproceedings{Vien2017ProbingTC, title={Probing the conformation of a conserved glutamic acid within the Cl− pathway of a CLC H+/Cl− exchanger}, author={Malvin Vien and Daniel Basilio and Lilia Leisle and Alessio Accardi}, booktitle={The Journal of general physiology}, year={2017} }