Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter.

Multiscale reactive molecular dynamics simulations are used to study proton transport through the central region of ClC-ec1, a widely studied ClC transporter that enables the stoichiometric exchange of 2 Cl(-) ions for 1 proton (H(+)). It has long been known that both Cl(-) and proton transport occur through partially congruent pathways, and that their exchange is strictly coupled. However, the nature of this coupling and the mechanism of antiporting remain topics of debate. Here multiscale simulations have been used to characterize proton transport between E203 (Glu(in)) and E148 (Glu(ex)), the internal and external intermediate proton binding sites, respectively. Free energy profiles are presented, explicitly accounting for the binding of Cl(-) along the central pathway, the dynamically coupled hydration changes of the central region, and conformational changes of Glu(in) and Glu(ex). We find that proton transport between Glu(in) and Glu(ex) is possible in both the presence and absence of Cl(-) in the central binding site, although it is facilitated by the anion presence. These results support the notion that the requisite coupling between Cl(-) and proton transport occurs elsewhere (e.g., during proton uptake or release). In addition, proton transport is explored in the E203K mutant, which maintains proton permeation despite the substitution of a basic residue for Glu(in). This collection of calculations provides for the first time, to our knowledge, a detailed picture of the proton transport mechanism in the central region of ClC-ec1 at a molecular level.