title: Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus.
abstract: The lactose transport protein (LacS) of Streptococcus thermophilus catalyzes the uptake of lactose in an exchange reaction with intracellularly formed galactose. The interactions between the substrate and the cytoplasmic and extracellular binding site of LacS have been characterized by assaying binding and transport of a range of sugars in proteoliposomes, in which the purified protein was reconstituted with a unidirectional orientation. Specificity for galactoside binding is given by the spatial configuration of the C-2, C-3, C-4, and C-6 hydroxyl groups of the galactose moiety. Except for a C-4 methoxy substitution, replacement of the hydroxyl groups for bulkier groups is not tolerated at these positions. Large hydrophobic or hydrophilic substitutions on the galactose C-1 alpha or beta position did not impair transport. In fact, the hydrophobic groups increased the binding affinity but decreased transport rates compared with galactose. Binding and transport characteristics of deoxygalactosides from either side of the membrane showed that the cytoplasmic and extracellular binding site interact differently with galactose. Compared with galactose, the IC(50) values for 2-deoxy- and 6-deoxygalactose at the cytoplasmic binding site were increased 150- and 20-fold, respectively, whereas they were the same at the extracellular binding site. From these and other experiments, we conclude that the binding sites and translocation pathway of LacS are spacious along the C-1 to C-4 axis of the galactose moiety and are restricted along the C-2 to C-6 axis. The differences in affinity at the cytoplasmic and extracellular binding site ensure that the transport via LacS is highly asymmetrical for the two opposing directions of translocation.
authors.affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: L M
authors.initials: LM
authors.lastname: Veenhoff
authors.forename: B
authors.initials: B
authors.lastname: Poolman
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D003593:Cytoplasm / Q000378:metabolism; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids / Q000378:metabolism; D013291:Streptococcus / Q000378:metabolism*; D027981:Symporters
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D007785:Lactose; D005690:Galactose