SignificanceSubstituted d-galactopyranosides, particularly those in the α configuration and/or with hydrophobic constituents at the anomeric position, bind to LacY with higher affinity than the physiological substrate lactose that has a β configuration. The structure of a conformationally restricted LacY mutant with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. Higher affinity, gained by nonspecific hydrophobic interaction of the nitrophenyl group, shows identical interaction at the key galactosyl moiety as in thio-digalactoside and so validates the highly specific, oriented set of hydrogen bonds with the key galactosyl moiety of substrates. Confirmation of galactose-specific binding interactions delineates a directional hydrogen-bonding network that couples the binding site to sites that are sensitive to protonation in the mechanism.The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.