Structure/function relationships in OxlT, the oxalate-formate transporter of oxalobacter formigenes. Assignment of transmembrane helix 11 to the translocation pathway.
OxlT, the oxalate:formate antiporter of Oxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 --> Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate.
Fu
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Department of Physiology, The Johns Hopkins University Medical School, Baltimore, Maryland 21205, USA.
Sarker
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RI
Abe
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Bolton
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Maloney
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D001426:Bacterial Proteins; D001692:Biological Transport; D002352:Carrier Proteins; D007202:Indicators and Reagents / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020560:Proteobacteria / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C099549:OxlT protein, bacteria