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

Abstract

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-.

Shared Molecular Mechanisms of Membrane Transporters

“…Interestingly, chlorideproton exchangers (ClCs) (6) were thought, until recently, to be an exception to this rule because transport seemed to be associated only with the local movements of side chains within a rigid protein scaffold (7)(8)(9). However, new data suggest that global structural transitions are also likely to be part of the ClC mechanism (10)(11)(12). It should be noted that small perturbations, such as mutations or the binding of small molecules, can turn some transporters into channels, presumably by stabilizing the states in which both the extracellular and intracellular gates are open (e.g., a double mutant of the Escherichia coli ClC transporter becomes a channel) (6,13,14).…”

A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl–/H+ transport cycle

“…A central question, therefore, is whether and how other protein conformational changes contribute to the CLC transport mechanism. In previous work, we used a spectroscopic approach to evaluate conformational changes in CLC-ec1, and we found that raising [H + ] (to protonate Glu ex ) caused conformational change in regions of the protein outside of the permeation pathway, up to ~20 Å away from Glu ex (Elvington et al, 2009;Abraham et al, 2015). Using a combination of biochemical crosslinking, double electron-electron resonance (DEER) spectroscopy, functional assays, and molecular dynamics (MD) simulations, we concluded that this H + -induced conformational state represents an "outward-facing open" state, an intermediate in the transport cycle that facilitates anion transport to and from the extracellular side (Khantwal et al, 2016).…”

Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl−/H+ transport cycle

“…A central question therefore is whether and how other 2 protein conformational changes contribute to the CLC transport mechanism. In previous work, we 3 used a spectroscopic approach to evaluate conformational changes in CLC-ec1, and we found that 4 raising [H + ] (to protonate Glu ex ) caused conformational change in regions of the protein outside of the 5 permeation pathway, up to ~20 Å away from Glu ex (Elvington et al, 2009;Abraham et al, 2015). 6…”