¹³C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

CLC transporters catalyze the exchange of Cl⁻ for H⁺ across cellular membranes. To do so, they must couple Cl⁻ and H⁺ binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state ¹³C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H⁺) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H⁺-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl⁻-permeation pathway, to the extracellular solution. The H⁺-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H⁺ binding is mechanistically coupled to closing of the intracellular access-pathway for Cl⁻