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

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, Cl - channels 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, Glu ex could explain the transport cycle of CLC-type Cl - /H+ antiporters. However, the presence of multiple conformations of the Glu ex 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 Glu ex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.