BINDING OF MONOCLONAL-ANTIBODY 4B1 TO HOMOLOGS OF THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

The conformationally sensitive epitope for monoclonal antibody (mAb) 4 B1, which uncouples lactose from H+ translocation in the lactose perme ase of Escherichia coli, is localized in the periplasmic loop between helices VII and Vm (loop VII/VIII) on one face of a short helical segm ent (Sun J, et al., 1996, Biochemistry 35:990-998). Comparison of sequ ences in the region corresponding to loop VII/VIII in members of Clust er 5 of the Major Facilitator Superfamily (MFS), which includes five h omologous oligosaccharide/H+ symporters, reveals interesting variation s. 4B1 binds to the Citrobacter freundii lactose permease or E. coli r affinose permease with resultant inhibition of transport activity. Bec ause E. coli raffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide b ackbone at this position. Consistently, E. coli lactose permease with Pro in place of Gly254 also binds 4B1. In contrast 4B1 binding is not observed with either Klebsiella pneumoniae lactose permease or E. coli sucrose permease. When the epitope is transferred from E. coli lactos e permease (residues 245-259) to the sucrose permease, the modified pr otein binds 4B1, but the mAb has no significant effect on sucrose tran sport. The studies provide further evidence that the 4B1 epitope is re stricted to loop VII/VIII, and that 4B1 binding induces a highly speci fic conformational change that uncouples substrate and H+ translocatio n.