Proteolytic mapping and substrate protection of the Escherichia coli melibiose permease.
The topology and substrate-induced conformational change(s) of the Na+ (Li+ or H+)-melibiose cotransporter (MelB) of Escherichia coli were investigated by limited protease digestion. To facilitate these analyses, MelB was epitope-tagged both at its carboxyl-terminus and at its amino-terminus. Limited digestion with different proteases indicates that the cytoplasmic loops connecting transmembrane domains 4-5, 6-7, and 10-11 together with the carboxyl-terminus of MelB are exposed in the cytoplasm. In contrast, periplasmic loops are highly resistant to all the proteases examined, including nonspecific proteases such as proteinase K and thermolysin. The effect of Na+ or Li+ and/or melibiose on the rate of protease digestion of the cytoplasmic loops was also analyzed. The rate of protease digestion of loop 4-5 is specifically reduced, by approximately 3-fold, by the presence of Na+ or Li+. These results suggest that loop 4-5 is near or part of the cation binding site. Moreover, the presence of both melibiose and either Na+ or Li+ further reduced the rate of protease digestion of this loop 4-5 by up to 9-fold, although no protection from protease digestion was observed when melibiose was added alone. The increase in resistance to proteases observed in the presence of the cation alone or the cation plus melibiose suggests that the interaction of the two cosubstrate with MelB results in change(s) of MelB conformation.
Gwizdek
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Laboratoire J. Maetz, Département de Biologie cellulaire et moléculaire du Commissariat à l'Energie Atomique, B.P. 68, 06238 Villefranche-sur-mer, France.
Leblanc
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Bassilana
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D008081:Liposomes / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology; D008553:Melibiose / Q000378:metabolism / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012964:Sodium / Q000494:pharmacology; D027981:Symporters
D000939:Epitopes; D008081:Liposomes; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D008094:Lithium; D012964:Sodium; D010450:Endopeptidases