Figure 1.
Ion transport pathways and solvent accessibility of CLC-ec1.
(a) CLC-ec1 (PDB #1OTS) is shown in surface view, with subunits of the homodimer differently colored and drawn to indicate different aspects of the antiport mechanism. Key mechanistic residues are space-filled. Bifurcated Cl− and H+ pathways are indicated as dashed lines on right subunit. Separation of Gluin from the intracellular solution is shown with a blue arrow, central region between Gluin and Gluex with a red arrow, and the internal and central Cl− ions as green spheres. In subsequent figures, the internal Cl− ion is omitted, since this binds weakly and is unlikely to be directly involved in the transport mechanism. (b) Close-up view of the intracellular surface of CLC-ec1 near Gluin. Aqueous clefts are shown as dots, and the twin subunit is shown in greyscale to visualize the subunit interface. Polar and interfacial pathways—possible routes for H+ access to Gluin—are indicated with arrows.
Figure 2.
Effect of E202 mutations on Cl−-driven H+ uptake.
(a) H+ uptake traces of E202A and E202Q. Transport is initiated by addition of Vln (arrow) and terminated by FCCP, and H+ uptake is indicated by upward deflection in the pH traces shown. (b) H+ uptake traces with indicated substitutions at E202. (c) Correlation of initial H+ uptake rate with side chain volume. (d) Cl− efflux traces with indicated substitutions at E202. Cl− appearance in the liposome suspension is normalized to final value after addition of β-octylglucoside detergent (after abrupt step in trace). Arrow indicates addition of Vln+FCCP. (e) Summary of initial rates of Cl− efflux, γo, for E202 substitutions tested.
Figure 3.
Effect of E202 substitutions on Cl− pathway.
(a, b) Equilibrium Cl− binding isotherms determined by ITC for wild type or E202Y, respectively. Solid curves represent single-site binding curves with KD = 0.74 and 1.6 mM, respectively (Table S3). (c) Effect of E202F mutation on Cl− efflux traces in fully H+-uncoupled transporter, E148A. For comparison, dashed lines reprise the effect of E202F on the H+-coupled wild type. (d) Summary of E202F effect on Cl− efflux rates γo on coupled (WT) and uncoupled (E148A) backgrounds. Rates are normalized to the background value for comparison.
Figure 4.
The mutant backbone is basically unaltered from wild type (Cα rmsd 0.9 Å). (a) Structural comparison between wild-type (ΔNC) and E202Y mutant near the E202 residue. Residues are colored in yellow (for wild type) or green (for E202Y), red (oxygen), and blue (nitrogen). Cytoplasmic side view into the apex region of the interfacial pathway for wild type (b) and E202Y (c). E202N and I201N indicate residues coming from the neighboring subunit of the homodimer. Crystallographic water molecules are shown in blue dots. 2Fo-Fc maps are contoured at 1.0 σ.
Figure 5.
Test of E202 mechanism in a monomeric transporter.
Effects of the E202Y mutation on transport were tested on a monomeric variant of CLC-ec1 in which a double-mutant (I201W/I422W) disrupts the homodimer interface [8]. Rigorous, complete monomer formation requires phosphatidylcholine/phosphatidylglycerol liposomes, in which transport rates are 2–4-fold slower than with E. coli phospholipids. (a) Representative H+ transport traces for WT and E202Y on the monomeric background construct. (b) Comparison of inhibitory effect of E202Y substitution on H+ uptake by dimeric versus monomeric transporters. (c) Cl−/H+ exchange stoichiometry (3.1, Table S2) for monomeric E202Y was determined from initial rates (dashed lines).
Figure 6.
Proposed mechanism of intracellular H+ access.
Cartoon depicts (a) homodimeric CLC-ec1, with subunits colored grey or white. In each subunit, Cl− (green spheres) and crystallographic water molecules (blue dots) are shown. The proposed water-mediated interfacial H+ pathway connecting bulk intracellular water to the protein interior is indicated by blue arrows. Also shown are Gluin (red sticks) and the serine, tyrosine, and extracellular glutamate residues that coordinate the central Cl−. (b) E202Y mutant, with its substituted side chain pointing out to the blocked interfacial pathway and recruiting I201N (N denotes residue of neighboring subunit). Stick thickness represents vertical location of side chains. The polar pathway is also indicated as capped by the E117-R209 salt bridge.