Three-dimensional structure of a bacterial oxalate transporter

Hirai, Takeshi; Heymann, Jürgen; Shi, Dan; Sarker, Rafiquel; Maloney, Peter C.; Subramaniam, Sriram

doi:10.1038/nsb821

Cited by 143 publications

(215 citation statements)

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“…By contrast, Hirae and Subramaniam (12) address a conformation in which OxlT has bound ligand. As a result, this earlier model focuses on aspects of GlpT helix packing that, after an appropriate rigidbody rotation of N-and C-terminal domains (see below), best fit the OxlT 6.5-Å structure (6,7). This finding led to the proposal that helix organization in the GlpT C-terminal domain (GlpT ) is the preferred scheme for both halves of OxlT (OxlT TM1-6 and OxlT TM7-12 ).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Yang

Wang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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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, ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Yang

Wang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Although speculative, an imaginary folded conformation of MdfA might bring together the two putative substrate recognition clusters to form part of a continuous pathway. According to a recently proposed structural model for 12-helix transporters from major facilitator superfamily (41,42), TM 1, 2, 4, 5, 7, 8, 10, and 11 form a central cavity, which serves as a pathway for substrate transport. It was suggested that different transporters might use different combinations of residues from one or more of these eight helices to achieve the required substrate recognition spectrum (42).…”

Section: Effect Of the Substrates On [ 14 C]nem Labeling Of Single-mentioning

confidence: 99%

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Adler

Bibi

2004

Journal of Biological Chemistry

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The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteineless MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (⌬˜H؉, inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.

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“…The advent of crystal structures of several MFS transporters (8)(9)(10)(11) has now placed functional studies of this flavor of transporter on a much firmer footing and allowed for a more rigorous analysis. One of these transporters, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) in the Escherichia coli inner membrane, transports G3P into the cell via an antiport mechanism that is energized by an inorganic phosphate (P i ) gradient (12).…”

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Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

et al. 2007

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Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rockerswitch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (K m ) nor the apparent substrate-binding dissociation constant (K d ) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol −1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward-and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.Secondary active membrane transporter proteins use an electrochemical gradient generated across the membrane by primary active transport as the driving force for substrate translocation (1,2). Over 100 families of secondary active membrane transporters have been identified to date, the largest of which is the major facilitator superfamily (MFS), 1 with over 5000 known members (3-5). The MFS, members of which are ubiquitous in all three kingdoms of life, includes many medically and pharmaceutically important transporters, such as the efflux pumps that confer resistance to antibiotics in bacteria and chemotherapeutic drugs in humans (6,7). † This work was supported in part by NIH Grant . The work of P.C.M. was supported by NIH Grant GM-24195. * To whom correspondence should be addressed. Telephone: (212) 263-8951. E-mail: wang@saturn.med.nyu.edu. ‡ New York University School of Medicine. § Johns Hopkins School of Medicine.1 Abbreviations: GlpT, glycerol-3-phosphate transporter; G3P, glycerol-3-phosphate; P i , inorganic phosphate; MFS, major facilitator superfamily; E a , activation energy; DDM, N-dodecyl-β-D-maltoside. Over the years, extensive efforts have been made to understand the molecular ...

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Three-dimensional structure of a bacterial oxalate transporter

Cited by 143 publications

References 20 publications

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

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