Structure/function relationships in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes: assignment of transmembrane helix 2 to the translocation pathway

The Journal of Biological Chemistry
Liwen Ye, Peter C Maloney

Abstract

We constructed a single cysteine panel encompassing transmembrane helix two (TM2) of OxlT, the oxalate/formate antiporter of Oxalobacter formigenes. Among the 21 positions targeted, cysteine substitution identified one (phenylalanine 59) as essential to OxlT expression and three (glutamine 56, glutamine 66, and serine 69) as potentially critical to OxlT function. By probing membranes with a bulky hydrophilic probe (Oregon Green maleimide) we also located a central inaccessible core of at least eight residues in length, extending from leucine 61 to glycine 68. Functional assays based on reconstitution of crude detergent extracts showed that of single cysteine mutants within the TM2 core only the Q63C variant was substantially (> or =95%) inhibited by thiol-specific agents (carboxyethyl methanethiosulfonate and ethylsulfonate methanethiosulfonate). Subsequent analytical work using the purified Q63C protein showed that inhibition by ethylsulfonate methanethiosulfonate was blocked by substrate and that the concentration dependence of such substrate protection occurred with a binding constant of 0.16 mm oxalate, comparable with the Michaelis constant observed for oxalate transport (0.23 mm). These findings lead us to conclude that p...Continue Reading

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Citations

Mar 27, 2013·Proceedings of the National Academy of Sciences of the United States of America·Dana YaffeShimon Schuldiner
Jul 27, 2011·PloS One·Roman K GóreckiJacek K Bardowski
Jun 4, 2005·Proceedings of the National Academy of Sciences of the United States of America·Qiang YangPeter C Maloney
Sep 17, 2014·The Journal of Membrane Biology·Osigbemhe IyalomheDi Cody Kang
Apr 12, 2005·Protein Engineering, Design & Selection : PEDS·Thijs Beuming, Harel Weinstein
Dec 8, 2004·Archives of Biochemistry and Biophysics·Drazenka SvedruzićNigel G J Richards

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