Design, function and structure of a monomeric ClC transporter

Abstract

Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual-the generation of the kilowatt pulses with which electric fish stun their prey-to the quotidian-the acidification of endosomes, vacuoles and lysosomes. The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch Cl(-) channel and later confirmed by crystal structures of bacterial Cl(-)/H(+) antiporters, is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins-the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl(-) and H(+) antiport in the transporters-are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent crosslinks across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity, we cannot declare as definitive a 'parallel-pathways' picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC Cl(-)/H(+) exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl(-)/H(+) exchange in ClC transporters.

Figure 1. Structure and dimeric interface of ClC-ec1

Left panel: ClC-ec1 dimer (PDB: 1OTS) is shown with subunits in grey and blue, with hydrophobic residues highlighted in yellow and tryptophan, tyrosine in magenta. Level of the membrane (extracellular side up) is indicated by black lines. Previously proposed transport pathways³¹ are shown for Cl⁻ and H⁺. Right panel: single subunit rotated 90^o to view the dimerization interface head-on. The four interface helices (residues 192–204, 215–232, 405–416, 422–440) are shown in red and sidechains involved in cross-subunit contacts in yellow.

Figure 2. Behavior of tryptophan mutants in detergent

a. A schematic of the dimerization interface showing positions of tryptophans tested. L194W did not express protein. b. Chromatographic profiles of the various mutants on Superdex 200. Vertical lines mark elution volumes for dimer and monomer. c. 10% SDS-PAGE of wildtype and WW samples, coomassie stained. Bars indicate samples at 0.25 mg/mL treated with 0.125% glutaraldehyde, 150 mM NaCl, 50 mM Na-phosphate pH 7.0 for the indicated times in 5 mM decylmaltoside or, as a negative control, in 2% SDS. Note that crosslinking is nearly complete in 1 minute, and that no higher oligomers appear even at 30 min.

Figure 3. Monomeric CLC mutant in phospholipid membranes

a. Glutaraldehyde crosslinking of wildtype ClC-ec1 and WW mutant in liposomes. Glutaraldehyde treatment was as in Fig 2, except that protein was incorporated into PC/PG liposomes, and gel was silver-stained. b. Passive Cl⁻ efflux from reconstituted liposomes for wildtype ClC-ec1 and WW mutant. Traces show release of Cl⁻ from liposomes loaded with 300 mM Cl⁻ into the extraliposomal solution (containing 1 mM Cl⁻) initiated by 0.5 μM valinomycin (downward arrow), normalized to the level of complete release upon disrupting liposomes with 50 mM octylglucoside (upward arrow). Unitary turnover calculated on a per-subunit basis from the initial rate of Cl⁻ release was: 290 ± 30 s⁻¹ for wildtype, 160 ± 9 s⁻¹ for WW (mean ± s.e.m, N=9). c. Cl⁻-driven H⁺ pumping against a pH gradient. Liposomes loaded with 300 mM Cl⁻ pH 5.0, were suspended in 1 mM Cl⁻, pH 5.2, and transport was initiated by valinomycin and terminated by FCCP (arrows), while pH of suspension was recorded. Upward deflection represents uptake of H⁺ into liposomes.

Figure 4. Crystal structure of WW monomer

a. View of two monomers in side-by-side contact, with interface helices highlighted in red, Cl⁻ ion in green, and additional symmetry-related monomers in the background (grey). b. Backbone alignment (Cα r.m.s.d. 0.6 X) of WW monomer (yellow, with interface helices in red) with a single subunit of wildtype CLC-ec1 (grey). Blue spheres indicate the N-termini of the visible structures. c. Central anion-binding site. 2Fo-Fc map (blue, 1.5 σ) is shown near the central Cl⁻-binding site, with coordinating residues Ser107, Glu148, and Tyr445 highlighted (yellow); positive difference density calculated from a Cl⁻-omit map (green) shows a strong peak (3.5 σ) at the position of the central Cl⁻ ion in wildtype. Stereo versions of panels a and c are displayed in Supplementary Fig 2.