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 position 63 lies on the OxlT translocation pathway. Our conclusion is strengthened by the finding that position 63, along with most other positions relevant to TM2 function, is found on a helical face that can be cross-linked to the pathway-facing surface of TM11 (Fu, D., Sarker, R. I., Bolton, E., and Maloney, P. C. (2001) J. Biol. Chem. 276, 8753-8760).
Highlights
The antiporter OxlT1 carries out the electrogenic exchange of divalent oxalate with monovalent formate, a reaction that underlies generation of the proton-motive force in the Gramnegative anaerobe Oxalobacter formigenes (1–3)
Our findings provide direct evidence supporting the idea that TM2 lies on the OxlT substrate translocation pathway and that this domain contributes residues critical to OxlT function
An immediate goal is to integrate the emerging structural information with ongoing functional studies. For this purpose we have focused on identification of helices likely to line the OxlT substrate translocation pathway, using criteria developed in earlier studies of UhpT, the sugar phosphate carrier of E. coli (10, 14, 15)
Summary
OxlT, oxalate/formate antiporter of O. formigenes; TM2, transmembrane helix 2; OGM, Oregon Green maleimide carboxylic acid; MTS, methanethiosulfonate; MTSCE, carboxyethyl MTS; MTSET, ethyltrimethylammonium MTS; MTSES, ethylsulfonate MTS; MOPS, morpholinepropanesulfonic acid. The two-dimensional projection map of OxlT reveals a single central cavity representing the substrate translocation pathway (4), but it is not yet possible to recognize the individual helices that border this pathway or to determine which among them contain substrate-binding elements. To address these issues two experimental strategies have been developed. Of the twelve OxlT transmembrane helices, TM2 and TM11 are the least hydrophobic (11, 12) and the most likely to specify residues that interact with oxalate (the hydrophilic substrate) In this respect, TM11 has been an attractive candidate for some time because it contains lysine 355, the only charged residue in the OxlT hydrophobic sector and a likely substrate-binding element. Our findings provide direct evidence supporting the idea that TM2 lies on the OxlT substrate translocation pathway and that this domain contributes residues critical to OxlT function
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