Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand.
Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a [(125)I]IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.
Sondej
Melissa
M
Howard Hughes Medical Institute, Department of Physiology and Immunology, University of California, Los Angeles, California 90095-1662, USA.
Vázquez-Ibar
José Luis
JL
Farshidi
Arta
A
Peterkofsky
Alan
A
Kaback
H Ronald
HR
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005454:Fluorescence Polarization; D005697:Galactosides / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D027981:Symporters
D029968:Escherichia coli Proteins; D005697:Galactosides; D007457:Iodine Radioisotopes; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C032242:crr protein, E coli; C021499:lactose permease; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D008239:Lysine; D003545:Cysteine