title: Immunity protein release from a cell-bound nuclease colicin complex requires global conformational rearrangement.
abstract: Nuclease colicins bind their target receptor BtuB in the outer membrane of sensitive Escherichia coli cells in the form of a high-affinity complex with their cognate immunity proteins. The release of the immunity protein from the colicin complex is a prerequisite for cell entry of the colicin and occurs via a process that is still relatively poorly understood. We have previously shown that an energy input in the form of the cytoplasmic membrane proton motive force is required to promote immunity protein (Im9) release from the colicin E9/Im9 complex and colicin cell entry. We report here that engineering rigidity in the structured part of the colicin translocation domain via the introduction of disulfide bonds prevents immunity protein release from the colicin complex. Reduction of the disulfide bond by the addition of DTT leads to immunity protein release and resumption of activity. Similarly, the introduction of a disulfide bond in the DNase domain previously shown to abolish channel formation in planar bilayers also prevented immunity protein release. Importantly, all disulfide bonds, in the translocation as well as the DNase domain, also abolished the biological activity of the Im9-free colicin E9, the reduction of which led to a resumption of activity. Our results show, for the first time, that conformational flexibility in the structured translocation and DNase domains of a nuclease colicin is essential for immunity protein release, providing further evidence for the hypothesis that global structural rearrangement of the colicin molecule is required for disassembly of this high-affinity toxin-immunity protein complex prior to outer membrane translocation.
authors.affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom.
authors.forename: Mireille
authors.initials: M
authors.lastname: Vankemmelbeke
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Housden
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Penfold
mesh_terms: D001665:Binding Sites; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D003087:Colicins; D004220:Disulfides; D011994:Recombinant Proteins; D003851:Deoxyribonucleases
keywords: Bacteriocin; Escherichia coli; colicin; disulfide bond; translocation
references.citation: Biochemistry. 2006 Aug 29;45(34):10199-207
references.pmid: 16922495
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13849-54
references.pmid: 16166265
references.citation: Biochemistry. 2008 Dec 2;47(48):12802-9
references.pmid: 18986168
references.citation: Nat Struct Biol. 2002 Jun;9(6):476-84
references.pmid: 12021774
references.citation: Mol Cell. 2001 Nov;8(5):1053-62
references.pmid: 11741540
references.citation: Eur J Biochem. 1992 Jul 15;207(2):687-95
references.pmid: 1633820
references.citation: J Bacteriol. 2008 Jun;190(12):4342-50
references.pmid: 18408035
references.citation: J Mol Biol. 1996 Aug 2;260(5):731-42
references.pmid: 8709151
references.citation: Nat Rev Microbiol. 2010 Dec;8(12):843-8
references.pmid: 21060316
references.citation: J Bacteriol. 2007 Jun;189(11):4217-22
references.pmid: 17416663
references.citation: PLoS Biol. 2013;11(2):e1001489
references.pmid: 23431269
references.citation: EMBO J. 2009 Sep 16;28(18):2846-57
references.pmid: 19696740
references.citation: Annu Rev Genet. 2012;46:209-31
references.pmid: 22934645
references.citation: Mol Microbiol. 2011 May;80(3):588-95
references.pmid: 21443527
references.citation: J Bacteriol. 1991 Sep;173(18):5639-47
references.pmid: 1885541
references.citation: J Bacteriol. 2004 Jul;186(14):4520-7
references.pmid: 15231784
references.citation: Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21412-7
references.pmid: 21098297
references.citation: EMBO J. 2008 Aug 6;27(15):2171-80
references.pmid: 18636093
references.citation: Biochemistry. 2003 Apr 15;42(14):4161-71
references.pmid: 12680770
references.citation: Microbiol Mol Biol Rev. 2007 Mar;71(1):158-229
references.pmid: 17347522
references.citation: J Biol Chem. 2009 Jul 10;284(28):18932-41
references.pmid: 19458090
references.citation: Biochemistry. 1995 Oct 24;34(42):13743-50
references.pmid: 7577966
references.citation: Science. 2013 Jun 28;340(6140):1570-4
references.pmid: 23812713
references.citation: J Mol Biol. 2002 May 3;318(3):787-804
references.pmid: 12054823
references.citation: J Biol Chem. 2007 Oct 26;282(43):31389-97
references.pmid: 17720814
references.citation: Trends Biochem Sci. 2001 Oct;26(10):624-31
references.pmid: 11590016
references.citation: PLoS Biol. 2013;11(2):e1001486
references.pmid: 23431267
references.citation: Biochemistry. 2005 Aug 30;44(34):11496-507
references.pmid: 16114886
references.citation: J Bacteriol. 2005 Jul;187(14):4900-7
references.pmid: 15995205
references.citation: Nat Struct Biol. 2003 Nov;10(11):948-54
references.pmid: 14528295
references.citation: J Biol Chem. 2012 Jul 6;287(28):23381-96
references.pmid: 22593569
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12353-8
references.pmid: 16894158
references.citation: PLoS One. 2012;7(9):e46656
references.pmid: 23029560