Active-site-directed photolabeling of the melibiose permease of Escherichia coli.

Covalent photolabeling of the melibiose permease (MelB) of Escherichia coli has been undertaken with the sugar analogue [(3)H]-p-azidophenyl alpha-D-galactopyranoside ([(3)H]-alpha-PAPG) with the purpose of identifying the domains forming the MelB sugar-binding site. We show that alpha-PAPG is a high-affinity substrate of MelB (K(d) = 1 x 10(-)(6) M). Its binding to or transport by MelB is Na-dependent and is competitively prevented by melibiose or by the high-affinity ligand p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG). Membrane vesicles containing overexpressed histidine-tagged recombinant MelB were photolabeled in the presence of [(3)H]-alpha-PAPG by irradiation with UV light (lambda = 250 nm). Eighty-five percent of the radioactivity covalently associated with the vesicles was incorporated in a polypeptide corresponding to MelB monomer. MelB labeling was completely prevented by an excess of melibiose or alpha-NPG during the assay. Radioactivity analysis of CNBr cleavage or limited proteolysis products of the purified [(3)H]-alpha-PAPG-labeled transporter suggests that several domains of MelB are targets for labeling. One of the labeled CNBr cleavage products is a peptide with an apparent molecular mass of 5.5 kDa. It is shown that (i) its amino acid sequence is that of the Asp124-Met181 domain of MelB (7.5 kDa), which includes the cytoplasmic loop 4-5 connecting helices IV and V, the hydrophobic helix V, and the outer loop connecting helices V-VI, and (ii) that Arg141 in loop 4-5 is the only labeled amino acid of this peptide. Labeling of loop 4-5 provides independent evidence that this specific domain plays a significant role in MelB transport. Comparison with the well-characterized equivalent domain of LacY suggests that sugar transporters with similar structure and substrate specificity may have conserved domains involved in sugar recognition.