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

By Sherwin J. Abraham, Ricky C. Cheng, Thomas A. Chew, Chandra M. Khantwal, Corey W. Liu, Shimei Gong, Robert K. Nakamoto and Merritt Maduke.

Published in Journal of Biomolecular NMR, 2015 Jan 29. [Epub ahead of print] PMID: 25631353. PMCID: 4398623. Link to publication page.

Project: Conformational Dynamics in the CLC Channel/Transporter Family | Core Facility: Membrane Protein Expression and Purification

Figure 2. CLC structure and conformational change compared to conformational change detected in other membrane transporters. a X-ray crystallographic structure of ClC-ec1 (pdb ID: 1OTS). Each identical subunit catalyzes the exchange of 2 Cl- for 1 H+. The H+ pathway bifurcates from the Cl− pathway, passing along Gluex and Gluin. Gluex is also a “gate” that occludes Cl- from the extracellular solution. An intracellular Cl- gate is formed by a Ser/Tyr pair (shown in spacefill), the latter residing on Helix R (dark green), a subject of study here. b Structural comparison of WT ClC-ec1 with the Gluex → Gln mutant (E148Q). Close-up of the Cl−-binding region in WT (left) and E148Q (right). In E148Q, the glutamine side chain is flipped up out of the anion-binding site and replaced by a Cl− ion (pdb ID: 1OTU). c Structural comparison of active transporters crystallized in different conformational states. Top left: Backbone overlay of ClC-ec1 WT (orange) and E148Q (blue) structures, 0.6 Å RMSD, illustrates the lack of global conformational change in the putative outward-facing conformational state. Bottom left: GltPh, a glutamate transporter homolog (Focke et al. 2013) in outward-facing (orange) and inward-facing (blue) states (pdb 4OYE and 4P19) (Verdon et al. 2014). Bottom middle: LacY, a member of the Major Facilitator Superfamily of transporters (Yan 2013) in occluded (orange) and inward-facing (blue) conformational states (pdb 4OAA and 2V8N) (Guan et al. 2007; Kumar et al. 2014). Right: Maltose transporter, a member of the ATP-binding cassette (ABC) transporter superfamily (Chen 2013) in inward-facing (orange) and occluded (blue) states (pdb 3FH6 and 2R6G) (Khare et al. 2009; Oldham et al. 2007)


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−.


Figure 2. Summary of positions isotope-labeled for NMR studies on ClC-ec1

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Figure 5. Identification of the H+-sensitive lysine residue.

Figure 6. Functional characterization of reductively methylated ClC-ec1.