Secondary water pore formation for proton transport in a ClC exchanger revealed by an atomistic molecular-dynamics simulation

Abstract

Several prokaryotic ClC proteins have been demonstrated to function as exchangers that transport both chloride ions and protons simultaneously in opposite directions. However, the path of the proton through the ClC exchanger, and how the protein brings about the coupled movement of both ions are still unknown. In this work, we use an atomistic molecular dynamics (MD) simulation to demonstrate that a previously unknown secondary water pore is formed inside an Escherichia coli ClC exchanger. The secondary water pore is bifurcated from the chloride ion pathway at E148. From the systematic simulations, we determined that the glutamate residue exposed to the intracellular solution, E203, plays an important role as a trigger for the formation of the secondary water pore, and that the highly conserved tyrosine residue Y445 functions as a barrier that separates the proton from the chloride ion pathways. Based on our simulation results, we conclude that protons in the ClC exchanger are conducted via a water network through the secondary water pore, and we propose a new mechanism for the coupled transport of chloride ions and protons. It has been reported that several members of ClC proteins are not just channels that simply transport chloride ions across lipid bilayers; rather, they are exchangers that transport both the chloride ion and proton in opposite directions. However, the ion transit pathways and the mechanism of the coupled movement of these two ions have not yet been unveiled. In this article, we report a new finding (to our knowledge) of a water pore inside a prokaryotic ClC protein as revealed by computer simulation. This water pore is bifurcated from the putative chloride ion, and water molecules inside the new pore connect two glutamate residues that are known to be key residues for proton transport. On the basis of our simulation results, we conclude that the water wire that is formed inside the newly found pore acts as a proton pathway, which enables us to resolve many problems that could not be addressed by previous experimental studies.

Figure 1

Structural representations of the E. coli ClC exchanger. Equilibrated structures of the ClC exchanger with the primary pore (A) and the secondary pore (B) are shown, and two structures are shown overlapped in C to compare the structural differences between A and B. Two chloride ions are represented as green spheres in A, and the pores predicted by the HOLE program are shown with a yellow transparent color in A and B. The structures in A and B are colored red and blue, respectively, in C. The D-, F-, and N-helices are shown by cartoon representation, and key residues are shown in stick representation. All of the snapshot figures in this work were created with PyMOL (http://www.pymol.org).

Figure 2

Distance and energy between E203(Q203) and R28 during the 14 ns simulation. Red, green, and blue lines represent the distance and energy between R28 and its pair (deprotonated E203, protonated E203, and Q203), respectively.

Figure 3

Structural representations of E. coli mutant ClC exchangers. Simulations were performed with mutants E203Q (A) and Y445F (B). The D-, F-, and N-helices are shown by cartoon representation, and key residues are shown in stick representation. Hydrogen bonding between Q203 and R28 is represented by red lines.

Figure 4

Schematic representation of the coupled movement of chloride ions and protons. The D-, F-, and N-helices are represented by gray cylinders. (A) Chloride ions are represented as green spheres, and protons are represented as yellow spheres. Protonation of E148 causes the primary pore to be opened, which results in chloride ion conduction. (B) After the chloride ions pass through the primary pore, the interaction between E203 and the R28 is broken due to the protonation of E203, resulting in the formation of a water network in the secondary pore. (C) The proton belonging to E148 is transferred to a water molecule inside the secondary pore, and subsequently the primary pore is closed by the deprotonation of E148. (D) The proton passes through the secondary pore via the water network.