An Outward-Facing Open Conformational State in a CLC Transporter

As secondary active transporters, CLCs harness energy stored in one ion concentration gradient (Cl- or H+) to pump the other ion against its electrochemical gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. Crystal structures suggest that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Here, we combine spectroscopy, cross-linking studies, crystallography, and computation to evaluate this simple model. Using 19F NMR and double electron-electron resonance (DEER) spectroscopy, we show that as [H+] is increased to enrich the outward-facing state, residues distant from Gluex move. Consistent with the functional relevance of this motion, a cross-link designed to inhibit the motions reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. Together, the results indicate the existence of a previously uncharacterized “outward-facing open” conformational state. A computational model based on the asymmetry exchange hypothesis for transporters with inverted-topology repeats (Forrest et al. 2008, PNAS) predicts such a state. To test the computational model, we monitored H+-induced structural changes using DEER spectroscopy on >20 sets of doubly labeled ClC-ec1 protein samples. Experimental distance distributions are compared with those derived from the ClC-ec1 X-ray structure and the model using restrained-ensemble molecular dynamics simulations (Roux et al. 2013, J Phys Chem B). Preliminary results show H+-dependent structural changes consistent with the computational model that can also be used to refine it. These results highlight the relevance of global structural changes in CLC function and provide the necessary foundation for understanding the Cl-/H+ coupling mechanism.