Ligand recognition by the lactose permease of Escherichia coli: specificity and affinity are defined by distinct structural elements of galactopyranosides.
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.
Sahin-Tóth
M
M
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.
Akhoon
K M
KM
Runner
J
J
Kaback
H R
HR
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
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