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-[C-14]ethylmaleimi de-labeling of the substrate-protectable Cys148 in the binding site. Alkyla tion of Cys148 is blocked by D-galactose with an apparent affinity of appro ximately 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, in dicating that these OH groups participate in essential interactions. Intere stingly, 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 subs trate 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-gal actopyranosides bind more strongly than galactose, supporting the notion th at the cyclic pyranose conformation is the bound form and that the anomeric configuration at C-l 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 significant ly influenced by the functional group at C-l and its orientation. Taken tog ether, 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, 9 802-9807] and indicate that specificity toward galactopyranosides is govern ed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while bin ding affinity can be increased dramatically by hydrophobic interactions wit h the nongalactosyl moiety.