title: Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes.
abstract: Using the x-ray structure of the glycerol 3-phosphate transporter (GlpT), we devised a model for the distantly related oxalate transporter, OxlT. The model accommodates all earlier biochemical information on OxlT, including the idea that Lys-355 lies on the permeation pathway, and predicts that Lys-355 and a second positive center, Arg-272, comprise the binding site for divalent oxalate. Study of R272K, R272A, and R272Q derivatives verifies that Arg-272 is essential, and comparisons with GlpT show that both anion transporters bind substrates within equivalent domains. In 22 single-cysteine variants in TM7 and TM8, topology as marked by accessibility to Oregon green maleimide is predicted by the model, with similar concordance for 52 positions probed earlier. The model also reconciles cross-linking of a cysteine pair placed near the periplasmic ends of TM2 and TM7, and retrospective study of TM2 and TM11 confirms that positions supporting disulfide trapping lie at a helical interface. Our work describes a pathway to the modeling of OxlT and other transporters in the major facilitator superfamily and outlines simple experimental tests to evaluate such proposals.
authors.affiliation: Department of Physiology, Johns Hopkins Medical School, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yang
authors.forename: Xicheng
authors.initials: X
authors.lastname: Wang
authors.forename: Liwen
authors.initials: L
authors.lastname: Ye
authors.forename: Mark
authors.initials: M
authors.lastname: Mentrikoski
authors.forename: Elham
authors.initials: E
authors.lastname: Mohammadi
authors.forename: Young-Mog
authors.initials: YM
authors.lastname: Kim
authors.forename: Peter C
authors.initials: PC
authors.lastname: Maloney
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D008301:Maleimides; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D020624:Oxalobacter formigenes / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D008301:Maleimides; D026901:Membrane Transport Proteins; C099549:OxlT protein, bacteria