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

MicrobiologyOpen

Mireille Vankemmelbeke¹  Nicholas G. Housden²  Richard James¹  Colin Kleanthous² 

[1] School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom;Department of Biochemistry, University of Oxford, Oxford, United Kingdom

关键词: Bacteriocin;    colicin;    disulfide bond;    Escherichia coli;    translocation;   

DOI  :  10.1002/mbo3.122

来源: Wiley

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.