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

Abstract

CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).

Fig. 1. CLC structure and conformational change compared to conformational change detected in other membrane transporters

a X-ray crystallographic structure of ClC-ec1 (pdb ID: 1OTS). Each identical subunit catalyzes the exchange of 2 Cl^- for 1 H⁺. The H⁺ pathway bifurcates from the Cl^- pathway, passing along Glu_ex and Glu_in. Glu_ex is also a “gate” that occludes Cl^- from the extracellular solution. An intracellular Cl^- gate is formed by a Ser/Tyr pair (shown in spacefill), the latter residing on Helix R (dark green), a subject of study here. b Structural comparison of WT ClC-ec1 with the Glu_ex → Gln mutant (E148Q). Left: Close-up of the Cl^--binding region in WT and E148Q. In E148Q, the glutamine side chain is flipped up out of the anion-binding site and replaced by a Cl^- ion (pdb ID: 1OTU). c Structural comparison of active transporters crystallized in different conformational states. Top left: Backbone overlay of ClC-ec1 WT (orange) and E148Q (blue) structures, 0.6 Å RMSD, illustrates the lack of global conformational change in the putative outward-facing conformational state. Bottom left: GltPh, a glutamate transporter homolog (Focke et al. 2013) in outward-facing (orange) and inward-facing (blue) states (pdb 4OYE and 4P19) (Verdon et al. 2014). Bottom middle: LacY, a member of the Major Facilitator Superfamily of transporters (Yan 2013) in occluded (orange) and inward-facing (blue) conformational states (pdb 4OAA and 2V8N) (Guan et al. 2007; Kumar et al. 2014). Right: Maltose transporter, a member of the ATP-binding cassette (ABC) transporter superfamily (Chen 2013) in inward-facing (orange) and occluded (blue) states (pdb 3FH6 and 2R6G) (Khare et al. 2009; Oldham et al. 2007).

Fig. 2. Summary of positions isotope-labeled for NMR studies on CLC-ec1

Structures are shown in stereo view, focused in on one subunit of the homodimer. a In a previous study, the five buried tyrosines were labeled with 3-fluorotyrosine (four solvent-exposed Tyr residues were mutated to Phe). Y445 and Y419 were identified as participating in H⁺-dependent conformational change (Elvington et al. 2009). b & c In this study, Met residues (b) were ¹³C labeled on their methyl groups and Lys residues (c) were post-translationally ¹³C-dimethylated. The unresolved N- and C-termini of ClC-ec1 contain one Met and 2 Lys residues that are not shown in these diagrams. Residues K30, K131, K271, K442, and K455, examined in mutagenesis studies here, are indicated.

Fig. 3. ¹H-¹³C HSQC spectra of ¹³C-Met labeled ClC-ec1

a Wildtype ClC-ec1 (homodimer, 100 kDa), methionine methyl region. b Monomeric ClC-ec1 (I201W/I422W, 50 kDa). c [H+]-dependence of ¹H-¹³C HSQC spectra: blue, pH 7.5; orange, pH 5.0; black, reversal to pH 7.5. The asterisk (*) and dagger (†) indicate the most clearly resolved crosspeaks that exhibit H⁺-dependency. d Partially deuterated (∼80%) monomeric ClCe-c1 with prolonged data collection. e Size exclusion chromatograms of ClC-ec1. Top WT ClC-ec1; Middle monomeric ClC-ec1 (mClC-ec1) (I201W/I422W); Bottom Δ6 mClC-ec1 (mutating six Met residues as follows: M38L, M65I, M228V, M276T, M330V, and M332L) yields a mix of dimer and monomer populations.

Fig. 4. ¹³C-Lys ClC-ec1

a¹H-¹³C HSQC spectra of methylated ClC-ec1 at pH 7.5 (left), pH 4.5 (middle) and reversed to pH 7.5 (right). The spectral window is zoomed in on the ¹³C methyl signals arising from the Lys side chains (with the ¹³C signal from the methylated N-terminus not shown). Changes in [H⁺] induce spectral shifts, the most notable being the peak splitting and shift of the peak at δ_H = 2.7 ppm (highlighted by the red boxes) at low pH. b pH titration of methylated ClC-ec1. Left: Overlaid ¹H-¹³C HSQC spectra at pH 7.5, 6.5, 6.0, 5.5, 5.0, 4.5, and 4.0, as indicated in the figure. Right: ¹H chemical shifts for the peak of interest as a function of pH. The solid curve is a fit to a single-site H⁺-binding isotherm, with an apparent pKa of 5.6. c¹H-¹³C HSQC spectra of methylated channel-like mutant at pH 7.5 (left), pH 4.5 (middle) and reversed to pH 7.5 (right). The spectrum of the channel-like mutant exhibits little [H⁺] sensitivity, and the peak at δ_H = 2.70 ppm and δ_C = 44.9 ppm is notably missing at both high and low pH.

Fig. 5. Identification of the H⁺-sensitive lysine residue

a¹H-¹³C HSQC spectra of methylated ClC-ec1 Lys-to-Arg mutants: K30R, K271R, K442R, and K455R. Unlike the channel-like mutant, these mutants maintain the spectral dispersion observed in WT ClC-ec1. Because Arg residues do not become ¹³C-methylated, these spectra allow assignment of the peak of interest (highlighted by the red boxes) to K455. b¹H-¹³C HSQC spectra of “K131 only”, a mutant in which all other Lys residues have been mutated to Arg. The minimal H⁺-dependence indicates that this region of the protein (Figure 2c) is not undergoing large H⁺-dependent conformational change.

Fig. 6. Functional characterization of reductively methylated ClC-ec1

a Schematic outline of the Cl^- flux assay. ClC-ec1 is reconstituted into liposomes at high [KCl], and then the extravesicular solution is exchanged for a low-[KCl] solution (replacing Cl^- with the impermeant isethionate, “X^-”). Bulk Cl^- efflux through ClC-ec1 is initiated by valinomycin (a K⁺ ionophore) and CCCP (a H⁺ ionophore) to dissipate the electrical potential. Extravesicular [Cl^-] is monitored using a Ag/AgCl electrode. b Representative raw data traces for WT ClC-ec1 (red) and control liposomes (containing no protein) (black dashes). At the end of each experiment, the detergent Triton X-100 was added to lyse the liposomes and release all of the Cl^-. c Summary data for reductively methylated (RM) proteins studied here. The relative turnover rates (normalized to WT unlabeled ClC-ec1) were calculated from the initial slope (Δ[Cl^-]/Δt) after addition of Vln/CCCP, and averages ± SEM (n=3 to 6) are reported.

Fig. 7. ¹³C-methyl thiocystine (MTC) ClC-ec1

a Position of helix R and I448. The position of helix R in one subunit of the ClC-ec1 homodimer is shown in green. The intracellular Cl^- gate residue Y445 (shown in spacefill) is the first residue of Helix R. ¹³C-MTC probes were introduced at positions I448 (shown in spacefill) and S464 (not shown; part of the unresolved C-terminus of ClC-ec1). ¹³C-K455 (spacefilled) was investigated by ¹³C-Lys methylation (Figure 4). b Activity assays on ¹³C-MTC ClC-ec1 variants, with Cl^- flux assays were performed as in Figure 6. Activity is normalized to WT ClC-ec1. Cys mutants were introduced into a cys-less template ClC-ec1 (Nguitragool and Miller 2007). Labeling at I448C and at S464C (with labeling efficiencies 93±5% and 89±3% respectively) has no effect on activity. Data represent averages ± SEM for n=8-11 replicates. c¹H-¹³C HSQC spectra of ¹³C-I448MTC ClC-ec1 at pH 7.5, 5.0, 4.75, 4.7, 4.5, and reversed to pH 7.5. At pH 7.5 and pH 5.0, the ¹³C-MTC probe exhibits a single peak (δ_H = 2.70 ppm). At pH 4.75 and pH 4.7, a second peak at δ_H = 2.36 ppm is additionally observed, and at pH 4.5 only this second peak remains. On reversal to pH 7.5, the spectrum reverts to the single peak at δ_H = 2.70 ppm. d¹H-¹³C HSQC spectra of ¹³C-I448MTC ClC-ec1 (left) and ¹³C-S464MTC (right) at pH 7.5 (blue), pH 4.5 (orange) and (for I448-MTC) reversed to pH 7.5 (black). S464 resides in the unresolved C-terminus of the protein. Its insensitivity to pH changes suggests that the peak shift in observed with I448 is likely due to structural changes in the protein rather than a general effect of pH.