The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin.
The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.
Zakharov
Stanislav D
SD
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Zhalnina
Mariya V
MV
Sharma
Onkar
O
Cramer
William A
WA
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; C109237:OmpF protein; D018272:Porins; C488855:ceiC protein, E coli