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.
Ambroise
Y
Y
Service des Molécules Marquées, Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, 91191 Gif sur Yvette Cedex, France.
Leblanc
G
G
Rousseau
B
B
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005697:Galactosides / Q000138:chemical synthesis / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009598:Nitrophenylgalactosides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D010782:Photolysis; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D027981:Symporters
D005697:Galactosides; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D027981:Symporters; C404842:p-azidophenyl galactopyranoside; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C021548:melibiose permease; D001120:Arginine