Water access points and hydration pathways in CLC H ⁺ /Cl ⁻ transporters

Abstract

CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl − has been established, the pathway of H + translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H + -translocation. An extended (0.4 μs) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, sitespecific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-Å hydrophobic region between the two known H + transport sites [E148 (Glu ex ) and E203 (Glu in )]. Our finding that the formation of these water wires requires the presence of Cl − explains the previously mystifying fact that Cl − occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H + transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H + and Cl − ions in CLC transporters. membrane transporters | membrane proteins | membrane exchangers | antiporters | coupling mechanism T he chloride channel (CLC) family (1, 2) includes both passive Cl − channels and secondary active H + -coupled Cl − transporters (3-8). The latter, also known as H + /Cl − exchangers, drive uphill movement of H + by coupling the process to downhill movement of Cl − or vice versa, thereby exchanging the two types of ions across the membrane at fixed stoichiometry (9). ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3)(4)(5)(6)(7)(8)(12)(13)(14)(15)(16)(17). Detailed structural and functional studies of 11,[18][19][20][21][22][23][24][25][26][27] have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl − permeation pathway and its lining residues (10,18,25) and established the role of E148, also known as Glu ex , as the extracellular gate for the Cl − pathway (9, 11).Although much less is known about the H + translocation pathway (and mechanism), experimental studies have provided key information ...

Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl–/H+ Exchanger ClC-ec1

“…The simulation system of Cl − -bound ClC-ec1 was adopted from our previous study. 29 The systems with the other anions were generated by replacing the Cl − at the S cen site with either F − , ${\mathrm{NO}}_3^{-}$ (N replacing Cl − ), or SCN − (C replacing Cl − ). All simulation systems included ClC-ec1 dimers, therefore providing two independent copies of the anion-bound systems to be examined (we note that the two subunits in the crystal structure of ClC-ec1 are not identical).…”

“…We additionally demonstrated the importance of the Cl − ion bound at the S cen site for stabilizing the water wires, thereby proposing a putative mechanism underlying the coupling of the two ions. 29 …”

ClC-5: Physiological role and biophysical mechanisms

“…5), intracellular protons must arrive at the S cen position, the position of E gat nearest to the intracellular side. The precise H + pathway is unknown but a relatively well conserved glutamate residue, the proton glutamate, E prot , located close to the intracellular solution, is probably involved in shuttling protons from the inside to S cen [87][88][89]. The proton glutamate is located off pore, marking a separate entry/exit pathway for intracellular protons [87].…”

13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

“…CLC antiporters catalyze the stoichiometric exchange of two Cl - for a single H + . X-ray crystallographic structures, together with experimentation and computation, have provided invaluable information on details of Cl - coordination, H + -transport pathways, and the existence of “gates” that occlude access of bound Cl - from the intracellular and extracellular solutions (Figure 1a) (Accardi et al 2006; Accardi and Miller 2004; Accardi et al 2005; Basilio et al 2014; Cheng and Coalson 2012; Dutzler 2006; Dutzler et al 2002; Dutzler et al 2003; Faraldo-Gomez and Roux 2004; Feng et al 2010; Han et al 2014; Jayaram et al 2008; Jayaram et al 2011; Ko and Jo 2010; Kuang et al 2007; Lim and Miller 2009; Lim and Miller 2012; Walden et al 2007; Zhang and Voth 2011). According to the alternating access mechanism for active transport, opening and closing of these gates must be tightly coupled to ion binding and unbinding events (Jardetzky 1966; Law et al 2008; Tanford 1983).…”