Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli.
Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance. Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants. Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism. Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation.
Howard Hughes Medical Institute, Department of Physiology, University of California Los Angeles 90095-1662, USA.
J
J
Sun
J
J
Wu
N
N
Carrasco
H R
HR
Kaback
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001360:Avidin / Q000378:metabolism; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D027981:Symporters
D000911:Antibodies, Monoclonal; D000939:Epitopes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D001360:Avidin; C021499:lactose permease; D007785:Lactose