Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl^-/H⁺ antiporter.

Abstract

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl^- channels and Cl^-/H⁺ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested 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 (<i>Up</i>, <i>Middle</i>, and <i>Down</i>) of the Glu_ex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from <i>Escherichia coli</i> and cmCLC from a thermophilic red alga, <i>Cyanidioschyzon merolae</i> Thus, we aimed to investigate further the heterogeneity of Glu_ex-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Glu_ex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the "<i>Midlow</i>" conformation. We also found that an extra anion can be located above the external Cl^--binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl^--binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Glu_ex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.