The central domain of colicin N possesses the receptor recognition site but not the binding affinity of the whole toxin

Biochemistry
L J EvansJ H Lakey

Abstract

Colicin N is a three-domain pore-forming colicin which kills enterobacterial cells following an initial binding to its receptor, the outer membrane porin OmpF. The receptor-binding domain of colicin N alone, and attached to the translocation domain, was overexpressed and purified using a hexahistidine tag. The receptor domain attached to the pore-forming domain was obtained by enzymatic digestion. Circular dichroism spectroscopy showed that the domains have structure in keeping with the known structure of colicin N. The receptor domain was stable, retaining both secondary and tertiary structure in 2 M guanidine hydrochloride and at low pH. It bound to both OmpF and PhoE porin-producing Escherichia coli with no toxicity and protected sensitive E. coli against intact colicin N toxicity at high domain/ colicin N ratios. Its in vitro affinity for OmpF, as determined by isothermal titration microcalorimetry, was found to be approximately 50-fold weaker than that of native colicin N. The receptor domain was readily out-competed by native colicin N in in vivo fluorescence assays which, coupled with its structural stability, suggests that its interaction with OmpF is one of weak, reversible binding. Since neither of the double domain c...Continue Reading

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Citations

Jun 11, 2002·Journal of Molecular Biology·Emily S CollinsGeoffrey R Moore
Nov 7, 1998·Biochimica Et Biophysica Acta·S HeyseH Vogel
Nov 25, 1998·Research in Microbiology·R el KouhenJ M Pagès
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Mar 10, 2007·Microbiology and Molecular Biology Reviews : MMBR·Eric CascalesDanièle Cavard
Jun 25, 2008·FEBS Letters·Oliver HechtGeoffrey R Moore
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May 14, 2020·The Journal of Biological Chemistry·Katarina Bartoš JansenColin Kleanthous
May 16, 2013·Molecular Microbiology·Christopher L JohnsonJeremy H Lakey
Dec 14, 2018·The Biochemical Journal·William A CramerS D Zakharov
Mar 5, 2014·Molecular Microbiology·Karen S Jakes
Feb 1, 2021·The Journal of Biological Chemistry·Katarina Bartoš JansenColin Kleanthous

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