title: Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage.
abstract: Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9(s-s)) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9(s-s), we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9(AF)) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.
authors.affiliation: School of Molecular Medical Sciences, Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University Park, Nottingham NG7 2RD, United Kingdom.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Mireille N
authors.initials: MN
authors.lastname: Vankemmelbeke
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Holland
authors.forename: David C
authors.initials: DC
authors.lastname: Walker
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Penfold
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D003087:Colicins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004765:Enteropeptidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C109237:OmpF protein; D018272:Porins; D004765:Enteropeptidase; D004229:Dithiothreitol