title: Ligand recognition by the lactose permease of Escherichia coli: specificity and affinity are defined by distinct structural elements of galactopyranosides.
abstract: Specificity of substrate recognition in lactose permease is directed toward the galactosyl moiety of lactose. In this study, binding of 31 structural analogues of D-galactose was examined by site-directed N-[(14)C]ethylmaleimide-labeling of the substrate-protectable Cys148 in the binding site. Alkylation of Cys148 is blocked by D-galactose with an apparent affinity of approximately 30 mM. Epimers of D-galactose at C-3 (D-gulose) and C-4 (D-glucose) or deoxy derivatives at these positions exhibit no binding whatsoever, indicating that these OH groups participate in essential interactions. Interestingly, the C-2 epimer alpha-D-talose binds almost as well as D-galactose, while 2-deoxy-D-galactose affords no substrate protection, indicating that nonstereospecific H-bonding at C-2 is required for stable binding. No substrate protection is detected with D-fucose, L-arabinose, 6-deoxy-6-fluoro-D-galactose, 6-O-methyl-D-galactose, or D-galacturonic acid, suggesting that the C-6 OH is an essential H-bond donor. Both alpha- and beta-methyl D-galactopyranosides bind more strongly than galactose, supporting the notion that the cyclic pyranose conformation is the bound form and that the anomeric configuration at C-1 does not contribute to substrate specificity. However, methyl or allyl alpha-D-galactopyranosides exhibit 60-fold lower apparent K(d)'s than D-galactose, demonstrating that binding affinity is significantly influenced by the functional group at C-1 and its orientation. Taken together, the observations confirm and extend the current binding site model [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] and indicate that specificity toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by hydrophobic interactions with the nongalactosyl moiety.
authors.affiliation: Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
authors.forename: M
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: K M
authors.initials: KM
authors.lastname: Akhoon
authors.forename: J
authors.initials: J
authors.lastname: Runner
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding; D013379:Substrate Specificity; D027981:Symporters
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D005690:Galactose