The last two cytoplasmic loops in the lactose permease of Escherichia coli comprise a discontinuous epitope for a monoclonal antibody.
Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M. By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope. Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition. Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants. Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect. The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.
Sun
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Li
J
J
Carrasco
N
N
Kaback
H R
HR
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001360:Avidin / Q000494:pharmacology; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013104:Spheroplasts / Q000378:metabolism; D027981:Symporters
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D001360:Avidin; D001710:Biotin; C021499:lactose permease