Helix Proximity in OxlT, the Oxalate:Formate Antiporter ofOxalobacter formigenes

Abstract

Experiments were designed to evaluate the proximity of transmembrane helices two (TM2) and eleven (TM11) in the tertiary structure of OxlT, the oxalate:formate exchange transporter of Oxalobacter formigenes. A tandem duplication of the Factor Xa protease cleavage site (IEGRIEGR) was inserted into the central cytoplasmic loop of an OxlT cysteine-less derivative in which an endogenous cleavage site had been eliminated by mutagenesis (R248Q). Using this host, double cysteine derivatives were constructed so as to pair one of seventeen positions in TM2 with one of four positions in TM11. Following treatment of membrane vesicles with Cu(II)(1,10-phenanthroline)₃, molecular iodine, orN,N′-o-phenylenedimaleimide, samples were exposed to Factor Xa, and disulfide bond formation was assessed after SDS-polyacrylamide gel electrophoresis by staining with antibody directed against the OxlT C terminus. In the absence of disulfide bond formation, exposure to Factor Xa revealed the expected C-terminal 22-kDa fragment, a result unaffected by the presence of reductant. By contrast, after disulfide formation, OxlT mobility remained at 35 kDa, and appearance of the 22-kDa fragment required addition of 200 mm dithiothreitol prior to electrophoresis. The four TM11 positions chosen for cysteine substitution lie on a helical face known to interact with substrate. Similarly, TM2 positions supporting disulfide trapping were also confined to a single helical face. We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of OxlT.