title: Interactions of TolB with the translocation domain of colicin E9 require an extended TolB box.
abstract: The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.
authors.affiliation: Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Hands
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Holland
authors.forename: Mireille
authors.initials: M
authors.lastname: Vankemmelbeke
authors.forename: Lauren
authors.initials: L
authors.lastname: Fraser
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Macdonald
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Penfold
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport / Q000502:physiology; D003087:Colicins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D003087:Colicins; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; C488859:immE9 protein, E coli; C062094:tolB protein, E coli