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

Liwen Ye, Peter C. Maloney

Research output: Contribution to journalArticle

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 (ge;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).

Original languageEnglish (US)
Pages (from-to)20372-20378
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number23
DOIs
StatePublished - Jun 7 2002

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formic acid
Oxalobacter formigenes
Antiporters
Oxalates
Cysteine
Glutamine
Facings
Substrates
Complex Mixtures
Phenylalanine
Sulfhydryl Compounds
Leucine
Detergents
Glycine
Serine
Assays
Substitution reactions
Membranes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure/function relationships in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes : Assignment of transmembrane helix 2 to the translocation pathway. / Ye, Liwen; Maloney, Peter C.

In: Journal of Biological Chemistry, Vol. 277, No. 23, 07.06.2002, p. 20372-20378.

Research output: Contribution to journalArticle

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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 (ge;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).",
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