Binding of Hydrophobic d-Galactopyranosides to the Lactose Permease of Escherichia coli

Abstract

Binding of alpha- and beta-D-galactopyranosides with different hydrophobic aglycons was compared using substrate protection against N-ethylmaleimide alkylation of single-Cys148 lactose permease. As demonstrated previously, methyl- or allyl-substituted alpha-D-galactopyranosides exhibit a 60-fold increase in binding affinity (K(D) = 0.5 mM), relative to galactose (K(D) = 30 mM), while methyl beta-D-galactopyranoside binds only 3-fold better. In the present study, galactopyranosides with cyclohexyl or phenyl substitutions, both in alpha and beta anomeric configurations, were synthesized. Surprisingly, relative to methyl alpha-D-galactopyranoside, binding of cyclohexyl alpha-D-galactopyranoside to lactose permease is essentially unchanged (K(D) = 0.4 mM), and phenyl alpha-D-galactopyranoside exhibits only a modest increase in binding affinity (K(D) = 0.15 mM). Nitro- or methyl-substituted phenyl alpha-D-galactopyranosides bind with significantly higher affinities (K(D) = 0.014-0.067 mM), and the strongest binding is observed with analogues containing para substituents. In contrast, D-galactopyranosides with a variety of large hydrophobic substituents (isopropyl, cyclohexyl, phenyl, o- or p-nitrophenyl) in beta anomeric configuration exhibit uniformly weak binding (K(D) = 1.0-2.3 mM). The results confirm and extend previous observations that hydrophobic aglycons of D-galactopyranosides increase binding affinity, with a clear predilection toward alpha-substituted sugars. In addition, the data suggest that the primary interaction between the permease and hydrophobic aglycons is directed toward the carbon atom bonded to the anomeric oxygen. The different positioning of this carbon atom in alpha- or beta-D-galactopyranosides thus may provide a rationale for the characteristic binding preference of the permease for alpha anomers.

A chemiosmotic mechanism of symport

“…4) that allows assignment of likely H-bond interactions with the protein, although interatomic distances are only estimates at a resolution of 3.5 Å. Specificity is directed toward the galactopyranoside ring, and α-galactosides bind with higher affinity than the β-anomers (14)(15)(16)(17). One galactopyranosyl ring of TDG stacks hydrophobically with Trp151 (helix V), confirming biochemical (18) and spectroscopic (19) findings.…”

Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease

“…All of the substrate specificity of LacY is directed towards the galactopyranosyl moiety of its substrates, and substituents on the anomeric position increase affinity by non-specific interactions. 28 The side-chains involved in LacY specificity are located in N-terminal six-helix bundle in helices I, IV, and V (Figure 2(a)), as identified by site-directed and Cys-scanning mutagenesis 29 and by the X-ray structure. 5 Arg144 (helix V) forms a bidentate H-bond with the C 4 -OH and C 3 -OH of the galactopyranosyl ring.…”

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

“…Biochemical studies (1)(2)(3)(4)(5)(6)(7) indicate that affinity and specificity are distinct properties determined by different interactions with LacY. Specificity is determined entirely by interactions with the galactopyranosyl ring, whereas affinity is better with α-than β-galactopyranosides (anomeric at C1) and can be increased dramatically by hydrophobic anomeric substituents with no effect on specificity.…”

“…p-Nitrophenyl-α-D-galactopyranoside (α-NPG) is a FRET acceptor from Trp-151 (37) and binds to LacY with ∼eight times higher affinity than the twofold symmetric TDG, ∼two orders of magnitude better than β-NPG, and ∼three orders of magnitude better than the physiological substrate lactose or the monosaccharide galactose (6,7,38,39). As we show here, the sidechain interactions with the galactopyranosyl moiety of α-NPG that provide specificity are almost identical to those described for TDG.…”