title: Topology and accessibility of the transmembrane helices and the sensory site in the bifunctional transporter DcuB of Escherichia coli.
abstract: C(4)-Dicarboxylate uptake transporter B (DcuB) of Escherichia coli is a bifunctional transporter that catalyzes fumarate/succinate antiport and serves as a cosensor of the sensor kinase DcuS. Sites and domains of DcuB were analyzed for their topology relative to the cytoplasmic or periplasmic side of the membrane and their accessibility to the water space. For the topology studies, DcuB was fused at 33 sites to the reporter enzymes PhoA and LacZ that are only active when located in the periplasm or the cytoplasm, respectively. The ratios of the PhoA and LacZ activities suggested the presence of 10 or 11 hydrophilic loops, and 11 or 12 α-helical transmembrane domains (TMDs). The central part of DcuB allowed no clear topology prediction with LacZ/PhoA fusions. The sites of DcuB accessible to the hydrophilic thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate (AMS) were determined with variants of DcuB that carried single Cys residues. After intact cells were labeled with the membrane-impermeable AMS, denatured cells were differentially labeled with the thiol reagent polyethylene-glycol-maleimide (PEGmal) and analyzed for a mass shift. From 35 positions 17 were accessible to AMS in intact bacteria. The model derived from topology and accessibility suggests 12 TMDs for DcuB and a waterfilled cavity in its central part. The cavity ends with a cytoplasmic lid accessible to AMS from the periplasmic side. The sensory domain of DcuB is composed of cytoplasmic loop XI/XII and a membrane integral region with the regulatory residues Thr396/Asp398 and Lys353.
authors.affiliation: Institute for Microbiology and Wine Research, Johannes Gutenberg-University, Becherweg 15, 55099 Mainz, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Bauer
authors.forename: Max J
authors.initials: MJ
authors.lastname: Fritsch
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
authors.forename: Gottfried
authors.initials: G
authors.lastname: Unden
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002462:Cell Membrane / Q000737:chemistry*; D003545:Cysteine; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000166:cytology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013267:Stilbenes / Q000378:metabolism; D013451:Sulfonic Acids / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C414535:DcuA dicarboxylate transporter, bacteria; D029382:Dicarboxylic Acid Transporters; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D013267:Stilbenes; D013451:Sulfonic Acids; C086256:dcuB protein, E coli; C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D003545:Cysteine; D005033:Ethylmaleimide