The structure and intrinsic activities of conserved STAS domains of the ubiquitous SulP/SLC26 anion transporter superfamily have until recently remained unknown. Here we report the heteronuclear, multidimensional NMR spectroscopy solution structure of the STAS domain from the SulP/SLC26 putative anion transporter Rv1739c of Mycobacterium tuberculosis. The 0.87-Å root mean square deviation structure revealed a four-stranded ␤-sheet with five interspersed ␣-helices, resembling the anti-factor antagonist fold. Rv1739c STAS was shown to be a guanine nucleotide-binding protein, as revealed by nucleotide-dependent quench of intrinsic STAS fluorescence and photoaffinity labeling. NMR chemical shift perturbation analysis partnered with in silico docking calculations identified solvent-exposed STAS residues involved in nucleotide binding. Rv1739c STAS was not an in vitro substrate of mycobacterial kinases or anti-factors. These results demonstrate that Rv1739c STAS binds guanine nucleotides at physiological concentrations and undergoes a ligand-induced conformational change but, unlike anti-factor antagonists, may not mediate signals via phosphorylation.

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.anion-binding ͉ disulfide trapping ͉ major facilitator superfamily ͉ membrane protein ͉ permeation pathway O xlT, the oxalate͞formate antiporter of Oxalobacter formigenes (1, 2), belongs to the major facilitator superfamily (MFS), a large and diverse collection encompassing 30-40% of known transporters and permeases (www.biology.ucsd.edu͞ϳmsaier͞ transport). The main biochemical mechanisms associated with transporters (uniport, antiport, and symport) may be found within the MFS, whose individual members display a broad catalog of substrate specificity, including simple sugars and amino acids, intermediary metabolites, and even neurotransmitters (3). All members of the MFS share an architectural theme in which a central loop connects two groups of (typically) six transmembrane ␣-helices. Moreover, the superfamily as a whole is characterized by a short motif (GXXXDK͞R) at the cytoplasmic ends of TM2 and TM8 (3, 4), suggesting that these two six-helix clusters derived from a common ancestor; indeed, at times one finds a clear sequence homology between the N-and C-terminal domains (4, 5).Insight into the structure of MFS transporters is presently limited. Helix organization, symmetry, and connectivity were established first for OxlT, in work based on a low-resolution (6.5 Å) structure obtained by electron crystallography (6, 7). Subsequently, higher resolution (3.2-3.5 Å) was achieved by x-ray crystallography of two other MFS members from Escherichia coli, the H ϩ ͞lactose symporter (LacY) and the phosphate͞glycerol 3-phosphate antiporter (GlpT) (8, 9). These latter achievements have prompted several recent attempts to use LacY or GlpT as structural templates for models of other systems (10-13). With this in mind and to provide a detailed perspective to guide further work, we selected the GlpT structure as a template for derivation of a homology model of OxlT, ...

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His 9 -tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni 2؉ -linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.The gram-negative bacterium Oxalobacter formigenes sustains a proton motive force by utilizing a "virtual" proton pump based on the transport and metabolism of oxalate. An electric potential (negative inside) arises from action of the antiporter, OxlT, which links inward transport of divalent oxalate to the outward flow of monovalent formate, the product of oxalate decarboxylation. The net inflow of a single negative charge is then phenomenologically linked to generation of a pH gradient (alkaline inside), because decarboxylation of oxalate consumes a single cytosolic proton. Together, these elements comprise the proton motive force used to drive ATP synthesis in this obligate anaerobe (3,14,20,32). Virtual pumps of equivalent construction have now been observed in a number of microorganisms (14,22,25).It is evident that OxlT occupies a central position in the cell biology of O. formigenes and that study of this transporter is relevant to several aspects of microbial physiology. Added interest in OxlT stems from recent work (8,9,26) suggesting that this protein may also serve as a useful model for biochemical study of other transporters. Accordingly, OxlT may contribute to an understanding of membrane transport at both a functional level and a mechanistic level.Hydropathy analysis of the OxlT amino acid sequence, along with other considerations, suggests the presence of 12 transmembrane segments (TM1 to TM12) (1), consistent with the presumed structure of most other members of the major facilitator superfamily (MFS) (30), the superfami...

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 (>95%) inhibited by thiol-specific agents (carboxyethyl methanethiosulfonate and ethylsulfonate methanethiosulfonate). Subsequent analytical work using the purified Q63C protein showed that inhibition by ethylsulfonate methanethio The antiporter OxlT 1 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). Although this aspect of bacterial cell biology merits further attention, current studies of OxlT are directed to the development of structural models following the success of electron crystallography, which has established a two-dimensional projection map for this protein (4). Such work may have wider significance because OxlT belongs to the major facilitator superfamily (5), the largest group of evolutionarily related antiporters, uniporters, and symporters (6).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. On the one hand, helix proximity is being examined by disulfide trapping in double cysteine variants (7). In addition and as reported here, selected helices are being subjected to biochemical tests to identify a domain(s) that lines the transport pathway (8 -10).Of the twelve OxlT transmembrane helices, TM2 and TM11 are the least hydrophobic (11, 12) and therefore 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. Recent work now confirms that TM11 lines the transport pathway and that a positive charge at position 355 is essential to OxlT function (9). By contrast, evidence suggesting that TM2 might line the OxlT pathway has been speculative, deriving largely from its unusually high content of polar residues (Fig. 1) because these may facilitate substrate binding via hydrogen bonding (11).The expe...