title: Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB-AEC2 ) of Escherichia coli.
abstract: Under phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 μM and 5.1 ± 0.3 μM, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable.
authors.affiliation: Division of Microbial Physiology, Humboldt-Universität zu Berlin, Unter den Linden 6, D-10099, Berlin, Germany.
authors.forename: Steven
authors.initials: S
authors.lastname: Wuttge
authors.forename: Martin
authors.initials: M
authors.lastname: Bommer
authors.forename: Franziska
authors.initials: F
authors.lastname: Jäger
authors.forename: Berta M
authors.initials: BM
authors.lastname: Martins
authors.forename: Sophie
authors.initials: S
authors.lastname: Jacob
authors.forename: Anke
authors.initials: A
authors.lastname: Licht
authors.forename: Frank
authors.initials: F
authors.lastname: Scheffel
authors.forename: Holger
authors.initials: H
authors.lastname: Dobbek
authors.forename: Erwin
authors.initials: E
authors.lastname: Schneider
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; C090796:ATP-binding protein, bacteria; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D004952:Esters; D005994:Glycerophosphates; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C585885:UgpA protein, E coli; C021688:UgpB protein, E coli; C083717:UgpC protein, E coli; C585886:UgpE protein, E coli; C029620:alpha-glycerophosphoric acid