Transient Water Chains Connecting the Cytoplasmic and Extracellular Glutamate Gates in CLC

Abstract

CLC-ec1, a bacterial member of the CLC family, exchanges chloride ions and protons across the cellular membrane. A key step of the transport cycle of CLC- ec1 is the transfer of protons between the extracellular and cytoplasmic gates, E148 and E203, respectively. These residues are 15 A apart, and in dearth of any intermediate titratable groups needed for proton shuttling. Hence, it is an open question as to how protons shuttle between the two gates. Proton hopping through water chains provides a possible mechanism in this regard, having been investigated in numerous computational studies, given the lack of experimental structural data on water. Two possible chains, either involving Y445 or side-chain rotation of E203, have been proposed based on searching algorithms and short molecular dynamics (MD) simulations. We herein propose another water chain characterized through extended (0.42 μs) MD simulations of CLC-ec1 dimer. The water chain forms frequently (once every 50-100 ns) but transiently (lasting for <1–2 ns). Neither Y445 nor the side-chain rotation of E203 is needed for the water chain. The presence of the water chain, however, coincides with significant side-chain conformational changes of F199 and F357 around the chloride-binding site and F208 and F219 at the dimer interface remote from the ion permeation pathway. We further performed a 0.25 μs simulation of monomeric CLC-ec1, which has been shown to be structurally identical to the dimer, but with a halved activity. We show that water chains don’t form as readily in the monomeric simulation as what was observed in the dimer simulation; side-chain conformations of F199, F357, F208 and F219 are also different from the dimer. Our study supports the idea that both local and long-range factors could be important for the CLC-ec1 activity.