Helix proximity in OxlT, the oxalate : formate antiporter of Oxalobacter formigenes - Cross-linking between TM2 and TM11

Experiments were designed to evaluate the proximity of transmembrane helice s two (TM2) and eleven (TM11) in the tertiary structure of Ox1T, the oxalat e-formate exchange transporter of Oxalobacter formigenes. A tandem duplicat ion of the Factor Xa protease cleavage site (IE-GRIEGR) was inserted into t he central cytoplasmic loop of an Ox1T 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(Il)(1,10-phenanthroline)(3), molecu lar iodine, or NN'-o-phenylenedimaleimide, samples were exposed to Factor X a, and disulfide bond formation was assessed after SDS-polyacrylamide gel e lectrophoresis by staining with antibody directed against the Ox1T C termin us. In the absence of disulfide bond formation, exposure to Factor Xa revea led the expected C-terminal 22-kDa fragment, a result unaffected by the pre sence of reductant. By contrast, after disulfide formation, Ox1T mobility r emained 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 wi th substrate. Similarly, TM2 positions supporting disulfide trapping were a lso confined to a single helical face. We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of Ox1T.