title: Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies.
abstract: Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.
mesh_terms: D000483:Alleles; D000596:Amino Acids / Q000032:analysis*; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis* / Q000235:genetics; D009154:Mutation
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins