Closure of the cytoplasmic gate formed by TM5 and TM11 during transport in the oxalate/formate exchanger from Oxalobacter formigenes

Abstract

OxlT, the oxalate/formate exchanger of Oxalobacter formigenes, is a member of the major facilitator superfamily of transporters. In the present work, substrate (oxalate) was found to enhance the reactivity of the cysteine mutant S336C on the cytoplasmic end of helix 11 to methanethiosulfonate ethyl carboxylate. In addition, S336C is found to spontaneously cross-link to S143C in TM5 in either native or reconstituted membranes under conditions that support transport. Continuous wave EPR measurements are consistent with this result and indicate that positions 143 and 336 are in close proximity in the presence of substrate. These two residues are localized within helix interacting GxxxG-like motifs (G₁₄₀LASG₁₄₄ and S₃₃₆DIFG₃₄₀) at the cytoplasmic poles of TM5 and TM11. Pulse EPR measurements were used to determine distances and distance distributions across the cytoplasmic or periplasmic ends of OxlT and were compared with the predictions of an inside-open homology model. The data indicate that a significant population of transporter is in an outside-open configuration in the presence of substrate; however, each end of the transporter exhibits significant conformational heterogeneity, where both inside-open and outside-open configurations are present. These data indicate that TM5 and TM11, which form part of the transport pathway, transiently close during transport and that there is a conformational equilibrium between inside-open and outside-open states of OxlT in the presence of substrate.

Figure 1

GlpT-based homology model of OxlT showing the transport lining helices, TM5 (tan) and TM11 (magenta) (PDB ID: 1ZC7). The approximate location of the Factor Xa cleavage site on the C-terminal end of the loop between TM6 and TM7 is indicated. The transporter is in a cytoplasmic open configuration where the Cα carbons of residues near the ends of TM5 and TM11 (S336 and S143) are separated by 21 Å. Other helices in the N-terminal domain are colored in cyan, and the remaining helices in the C-terminal domain are in light green. Both the C- and N-termini are located on the cytoplasmic side of the membrane.

Figure 2

MTSCE inhibition of transport for S143C and S336C. Purified single-cysteine OxlT mutants were reconstituted into liposomes to form proteoliposomes under conditions where they were loaded with nonradiolabeled (cold) oxalate. The proteoliposomes were washed on a filter, treated with MTSCE, washed again, and assayed for ¹⁴[C]oxalate uptake. As MTSCE concentrations are increased, there is a loss in ¹⁴[C]oxalate uptake for (a) S336C and (c) S143C in the absence of any external nonradiolabeled oxalate. In a second experiment, the proteoliposomes were washed, incubated with MTSCE sufficient to produce a 50% inhibition in transport and simultaneously with increasing concentrations of external cold oxalate, washed again, and then assayed for ¹⁴[C]oxalate transport. The residual ¹⁴[C]oxalate transport as a function of external cold oxalate concentration is shown for (b) S336C and (d) S143C. Each data point is the average of three measurements.

Figure 3

Western blot demonstrating cross-linking of S143C/S336C in native vesicles. Native membrane vesicles (see Experimental Procedures) containing OxlT with the S143C/S336C mutations and a tandem Factor Xa cleavage site were treated with 200 μM copper(II)(1,10-phenanthroline)₃(CuPhe), 200 μM MTS-1-MTS (M1M), or 200 μM MTS-2-MTS (M2M) for 10 min and immediately quenched with 5 mM NEM and 10 mM EDTA. Washed vesicles were processed for western blotting and probed with a C-terminal polyhistidine antibody. Xlink indicates lanes with the addition of the indicated cross-linking agents; Factor Xa indicates lanes where the central loop of OxlT has been cleaved; DTT indicates lanes where DTT is used to reduce disulfides after Factor Xa cleavage. The positions of the covalently intact protein (OxlT), the C-terminal OxlT fragment (C-ter), and OxlT oligomer (OxlTn) are indicated.

Figure 4

Cross-linking of OxlT S143C/S336C and transport in liposomes. E. coli vesicles containing OxlT with S143C/S336C and a tandem Factor Xa cleavage site were subjected to an oxidizing agent or buffer alone prior to reconstitution into oxalate-loaded vesicles of E. coli polar lipids. (a) Proteoliposomes were immediately quenched with 5 mM NEM after reconstitution and processed for western blotting using a polyhistidine antibody. (b) Another aliquot was assayed for ¹⁴[C]oxalate transport in the presence of an oxidizing agent without (○) and with (●) the addition of DTT and in the absence of cross-linking agent without (△) and with (▲) DTT. Each point was measured in triplicate.

Figure 5

Effect of oxalate on cross-linking of S143C/S336C FXa. OxlT in native vesicles with S143C/S336C and a tandem Factor Xa cleavage site was treated with the oxidizing agent copper(II) phenanthroline. Cross-linking was followed on ice by quenching the samples at specific times by adding them to tubes preloaded with 5 mM N-ethyl maleimde and 10 mM EDTA, followed by cleavage with Factor Xa. (a) Western blots using a polyhistidine antibody, which show cross-linking of OxlT where buffer (20 mM Tris·HCl (pH 7.5)) was on the vesicle interior and either 10 mM potassium sulfate (left) or 10 mM potassium oxalate (right) were on the vesicle exterior. (b) Buffer on the vesicle interior was replaced with 10 mM potassium sulfate (left) or 10 mM potassium oxalate (right) , and either 10 mM potassium sulfate (left) or 10 mM potassium oxalate (right) are on the exterior. In panel a, ON refers to overnight incubation at 4 °C.

Figure 6

X-band CW EPR line shapes of MTSL-labeled OxlT. The spin-labeled side chain R1 was derivatized to double mutants as well as pairs of the corresponding single-cysteine mutants of OxlT, and X-band EPR spectra were recorded at room temperature. Spectra are shown for the interacting spin pairs (blue traces) and the equivalent spectra without dipolar interactions (black traces). The non-interacting spectra are obtained from the sum of the single-labeled spectra. (a, b) Spectra for S143R1/S336R1 in bilayers and DDM, respectively, where the simulated spectrum obtained from the distance distribution is shown (dashed red trace). The sample in panel a yielded a mean distance of 7.8 Å with a standard deviation of 0.3 Å from three independent experiments. (c, d) Spectra for L141R1/I338R1, which do not show evidence for strong dipolar interaction, for bilayer and DDM environments, respectively. The scans shown are 150 G. Distances were determined using a Fourier deconvolution approach implemented in the LabVIEW program Short Distances (provided by Christian Altenbach, UCLA).

Figure 7

Distances and distance distributions measured by double electron–electron resonance (DEER) for several spin pairs in OxlT. (a) Measurements across the periplasmic end of OxlT. The background-corrected DEER data is shown on the left (black traces) along with the fits to the data using a model-free approach (red traces). The distributions obtained are shown on the right, and an error range is indicated by the vertical error bars (shaded in gray) in the distribution. This error range is based on uncertainty in the background subtraction and dimensionality in the background form factor that produces fits within 15% of the RMSD of the best fits. These errors were obtained using the validation routine in DeerAnalysis. Shown in magenta are predictions of the distances and distance distributions based on the OxlT homology model using the PyMOL plug-in mtsslWizard. (b) Measurements across the cytoplasmic end of OxlT. (c) Homology model of OxlT along with the labeled sites used for DEER. Raw DEER data were processed and analyzed using the Matlab software package DEER Analysis.