Early crystal structures of prokaryotic CLC proteins identified three Cl - binding sites: internal (S int), central (S cen), and external (S ext). A conserved external GLU (GLU ex) residue acts as a gate competing for S ext. Recently, the first crystal structure of a eukaryotic transporter, CmCLC, revealed that in this transporter GLU ex competes instead for S cen. Here, we use molecular dynamics simulations to investigate Cl - transport through CmCLC. The gating and Cl -/H + transport cycle are inferred through comparative molecular dynamics simulations with protonated and deprotonated GLU ex in the presence/absence of external potentials. Adaptive biasing force calculations are employed to estimate the potential of mean force profiles associated with transport of a Cl - ion from S ext to S int, depending on the Cl - occupancy of other sites. Our simulations demonstrate that protonation of GLU ex is essential for Cl - transport from S ext to S cen. The S cen site may be occupied by two Cl - ions simultaneously due to a high energy barrier (∼8 Kcal/mol) for a single Cl - ion to translocate from S cen to S int. Binding two Cl - ions to S cen induces a continuous water wire from S cen to the extracellular solution through the side chain of the GLU ex gate. This may initiate deprotonation of GLU ex, which then drives the two Cl - ions out of S cen toward the intracellular side via two putative Cl - transport paths. Finally, a conformational cycle is proposed that would account for the exchange stoichiometry. © 2012 Biophysical Society.
Cheng, M. H., & Coalson, R. D. (2012). Molecular dynamics investigation of Cl - and water transport through a eukaryotic CLC transporter. Biophysical Journal, 102(6), 1363–1371. https://doi.org/10.1016/j.bpj.2012.01.056