Monitoring Substrate-Driven Structural Changes in a CLC Chloride-Proton Antiporter with Double Electron-Electron Resonance Spectroscopy

Abstract

ClC-ec1, a prokaryotic Cl-/H+ antiporter in the CLC family, has been crystallized under many conditions. Yet, aside from local structural differences at the chloride binding site, only one major conformation is observed. This failure of X-ray crystallography to reveal the unknown conformational states in the transport cycle motivates the use of alternative approaches. Using 19F-NMR, a previous study demonstrated that a tyrosine residue > 20 A from the chloride-transport pathway undergoes antiport-specific structural changes (Elvington et al. 2009. EMBO J). We combine computational and experimental approaches to further investigate possible protein movements during the antiport cycle. A computational model of ClC-ec1 based on the inverted topology repeat hypothesis (Forrest et al. 2008. PNAS) predicts structural changes that alter the accessibility of pathways to the central Cl- and H+ binding sites. To test the model, we introduced nitroxide paramagnetic spin labels to the ClC-ec1 homodimer via site-directed spin labeling and monitored structural changes using double electron-electron resonance (DEER) spectroscopy. Preliminary results are consistent with the inverted-topology repeat model and suggest that the antiport cycle involves structural changes beyond local movement at the chloride-binding site.