Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpBAEC2) of Escherichia coli

Authors

  • Steven Wuttge,

    1. Division of Microbial Physiology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author
    • Contributed equally to this work.
  • Martin Bommer,

    1. Division of Structural Biology and Biochemistry, Department of Biology, Humboldt-Universität zu Berlin, Berlin, Germany
    2. Macromolecular Crystallography Group, Institute of Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany
    Search for more papers by this author
    • Contributed equally to this work.
  • Franziska Jäger,

    1. Division of Microbial Physiology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author
  • Berta M. Martins,

    1. Division of Structural Biology and Biochemistry, Department of Biology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author
  • Sophie Jacob,

    1. Division of Microbial Physiology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author
  • Anke Licht,

    1. Division of Microbial Physiology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author
  • Frank Scheffel,

    1. Division of Microbial Physiology, Humboldt-Universität zu Berlin, Berlin, Germany
    Current affiliation:
    1. Milchwirtschaftliche Lehr- und Untersuchungsanstalt Oranienburg, Oranienburg, Germany
    Search for more papers by this author
  • Holger Dobbek,

    1. Division of Structural Biology and Biochemistry, Department of Biology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author
  • Erwin Schneider

    Corresponding author
    • Division of Microbial Physiology, Humboldt-Universität zu Berlin, Berlin, Germany
    Search for more papers by this author

For correspondence. E-mail erwin.schneider@rz.hu-berlin.de; Tel. (+49) 30 2093 8121; Fax (+49) 30 2093 8126.

Summary

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 MalFGUgpC could be functionally reconstituted while a UgpAEMalK complex was unstable.

Ancillary