Molecular dynamics investigation of the interaction between central-site chloride binding and proton transport in <i>Ec</i> CLC

The <i>Ec</i>CLC protein functions as a transporter that concurrently translocates chloride ions and protons. Although extensive experimental and theoretical studies have been conducted, a comprehensive understanding of the transport processes of chloride ions and protons, as well as their coupling mechanisms, remains incomplete. To elucidate these mechanisms, we constructed an equilibrated <i>Ec</i>CLC-membrane-solution system and monitored the process of water molecules entering the proton channel. Furthermore, three distinct conformations of protonated Glu_ex-"up", "out", and "down"-were identified during molecular dynamic simulations. In all conformations of protonated Glu_ex, continuous water chains composed of 3-6 water molecules bridge Glu_ex and Glu_in within the proton channel through hydrogen-bond networks, with an average water-chain formation rate of 4.18 ± 0.75 ns^-1. To assess the interaction between chloride and proton transport, we computed the potential of mean force (PMF) for chloride permeation through the ion channel under both water-containing and water-depleted conditions within the proton transport pathway. The results indicate that the presence of water molecules markedly lowers the energy barriers associated with chloride translocation. Additionally, the structural changes in the proton channel in the presence or absence of a chloride ion at the central binding site were compared during molecular dynamics simulations. The results indicate that following chloride ion dissociation from the central binding site, the proton channel radius expands, the number of water molecules within the channel increases, and the length of the water chain bridging Glu_ex and Glu_in increases.