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Immunity protein release from a cell-bound nuclease colicin complex requires global conformational rearrangement

Vankemmelbeke, Mireille; Housden, Nicholas G.; James, Richard; Kleanthous, Colin; Penfold, Christopher N.

Authors

Mireille Vankemmelbeke

Nicholas G. Housden

Richard James

Colin Kleanthous

Christopher N. Penfold



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.

Journal Article Type Article
Publication Date Oct 1, 2013
Journal Microbiology Open
Electronic ISSN 2045-8827
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 2
Issue 5
APA6 Citation Vankemmelbeke, M., Housden, N. G., James, R., Kleanthous, C., & Penfold, C. N. (2013). Immunity protein release from a cell-bound nuclease colicin complex requires global conformational rearrangement. Microbiology Open, 2(5), doi:10.1002/mbo3.122
DOI https://doi.org/10.1002/mbo3.122
Publisher URL http://onlinelibrary.wiley.com/doi/10.1002/mbo3.122/abstract;jsessionid=3A2D705786B7B9590449ABA7DAFA73E2.f03t01
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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