Contributed equally to this work.
Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB–AEC2) of Escherichia coli
Article first published online: 27 SEP 2012
© 2012 Blackwell Publishing Ltd
Volume 86, Issue 4, pages 908–920, November 2012
How to Cite
Wuttge, S., Bommer, M., Jäger, F., Martins, B. M., Jacob, S., Licht, A., Scheffel, F., Dobbek, H. and Schneider, E. (2012), Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB–AEC2) of Escherichia coli. Molecular Microbiology, 86: 908–920. doi: 10.1111/mmi.12025
- Issue published online: 9 NOV 2012
- Article first published online: 27 SEP 2012
- Manuscript Accepted: 30 AUG 2012
- Deutsche Forschungsgemeinschaft. Grant Numbers: SCHN 274/15-1, EXC 314
- Humboldt-Universität zu Berlin
- DFG. Grant Number: MA3348/2-1
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.