Molecular Dynamics Investigation of Cl- and Water Transport through a Eukaryotic CLC Transporter

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 (similar to 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.