Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage.

Journal of Bacteriology
Ying ZhangChristopher N Penfold

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, oxidi...Continue Reading

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Citations

Nov 17, 2011·Quarterly Reviews of Biophysics·Grigorios PapadakosColin Kleanthous
Jun 28, 2011·The Journal of Biological Chemistry·Mathieu ChauleauMiklos de Zamaroczy
May 18, 2012·The Journal of Biological Chemistry·Silke I PatzerKornelius Zeth
May 22, 2009·The Journal of Biological Chemistry·Mireille VankemmelbekeRichard James
Nov 8, 2008·Journal of Biochemistry·Denis DuchéRoland Lloubès
Sep 1, 2012·Annual Review of Genetics·Karen S Jakes, William A Cramer
Aug 22, 2009·The EMBO Journal·Daniel A BonsorColin Kleanthous
Sep 17, 2013·MicrobiologyOpen·Mireille VankemmelbekeChristopher N Penfold
Apr 22, 2014·Biochimica Et Biophysica Acta·Young Chan KimChristopher N Penfold
Sep 13, 2016·PloS One·Nasim NikkhooKaren L Maxwell

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