Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Abstract

Using the x-ray structure of the glycerol 3-phosphate transporter (GlpT), we devised a model for the distantly related oxalate transporter, OxlT. The model accommodates all earlier biochemical information on OxlT, including the idea that Lys-355 lies on the permeation pathway, and predicts that Lys-355 and a second positive center, Arg-272, comprise the binding site for divalent oxalate. Study of R272K, R272A, and R272Q derivatives verifies that Arg-272 is essential, and comparisons with GlpT show that both anion transporters bind substrates within equivalent domains. In 22 single-cysteine variants in TM7 and TM8, topology as marked by accessibility to Oregon green maleimide is predicted by the model, with similar concordance for 52 positions probed earlier. The model also reconciles cross-linking of a cysteine pair placed near the periplasmic ends of TM2 and TM7, and retrospective study of TM2 and TM11 confirms that positions supporting disulfide trapping lie at a helical interface. Our work describes a pathway to the modeling of OxlT and other transporters in the major facilitator superfamily and outlines simple experimental tests to evaluate such proposals.

Fig. 1.

OxlT homology model. (A) OxlT topology. Boxes show transmembrane helices terminating in loops whose beginning and ending residue numbers are indicated; the MFS signature is noted in red. OxlT cysteines (Cys-28 and Cys-271) are shown by enlarged circles, as are the active-site residues, Arg-272 and Lys-355. Also emphasized are two residues (Gln-63 and Ser-359) known to lie on the permeation pathway. Solid lines indicate membrane thickness as reported for GlpT. (B) Ribbon tracing of the OxlT model, as viewed from the lipid phase, with the N-terminal asparagine (residue no. 2) indicated in yellow for reference. The extended side chains of the OxlT ligand-binding residues, Arg-272 and Lys-355, are shown in red. For comparison, ligand-binding residues in GlpT (Arg-46 and Arg-269) are shown in green. (C) OxlT as viewed from the cytoplasm and showing the active-site residues noted in B. In both OxlT and GlpT, these active-site residues are separated by 10 Å.

Fig. 2.

R272 is required for OxlT function. [¹⁴C]Oxalate transport by oxalate-loaded proteoliposomes for the indicated variants is shown. (Inset A) An immunoblot, developed by using antibody directed against the OxlT C-terminal polyhistidine tag, indicates reconstitution of comparable amounts of parental and R272K proteins and ≈2-fold higher recoveries for the R272A and R272Q derivatives. (Insets B and C) One of four experiments comparing the kinetics of [¹⁴C]oxalate exchange by parental and R272K proteins (see text).

Fig. 3.

Accessibility to OGM. (A) Membranes from cells expressing the indicated single-cysteine derivatives were exposed to OGM, after which detergent-solubilized OxlT was purified and processed for SDS/PAGE. The fluorescence profile of the gel identifies proteins modified by OGM (Upper); Commassie brilliant blue staining of the same gel reveals total protein (Lower). Note that SDS/PAGE yields OxlT monomers, dimers, and oligomers (nmers), as indicated, because of an increased tendency for aggregation of mutants placed in the cysteine-less background (19). (B) Results of A mapped on the OxlT homology model as viewed from the lipid phase, together with 52 cases reported earlier and assigned to the cytoplasmic or periplasmic surfaces or to the hydrophobic core (22, 23). Coding with yellow and red indicates, respectively, residues that are or are not susceptible to in situ labeling by OGM. (C) Data from B as viewed from the lipid phase with the N terminus closest to the reader.

Fig. 4.

Disulfide trapping at the OxlT periplasmic surface. (A) Membranes from cells expressing the indicated double-cysteine derivatives were exposed to copper phenanthroline to initiate disulfide trapping. After quenching the reaction, samples were incubated with or without Factor Xa protease and processed for SDS/PAGE (21), with reductant added before electrophoresis where indicated. Immunoblots were developed by using antibody directed against the OxlT C-terminal polyhistidine tag. (B) The OxlT model as viewed from the periplasmic surface (N terminus at the left). The residue pair that supports cross-linking (nos. 49 and 242) is shown in green; residue no. 240 is indicated in red. (C) Retrospective analysis of disulfide trapping in TM2 and TM11, using the ribbon diagram of Fig. 1B to display the target helices; TM2 residues highlighted in green cross-link with at least one of four residues in TM11 (shown in yellow), whereas TM2 residues indicated in red did not engage in cross-linking (21).