Abstract
Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low-copy-number, high-affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high-affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor-binding domain (ColN-R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid- and solid-state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS(Δ) (LIPID) ) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.
Highlights
Colicins are a family of highly effective bactericidal proteins which are produced by, and toxic to, related strains of Escherichia coli (E. coli)
Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell’s outer membrane barrier to kill
Genetic screens have suggested that colicin N (ColN), which has no highaffinity receptor, targets highly abundant lipopolysaccharide (LPS) instead
Summary
Colicins are a family of highly effective bactericidal proteins which are produced by, and toxic to, related strains of Escherichia coli (E. coli) They consist of three functional domains, an N-terminal translocation (T) domain, a central receptor binding (R) domain and a C-terminal domain which carries the lethal activity (Fig. 1A). ColE2 and E3 have similar R-domain sequences, possessing an extended ∼ 100 Å coiled-coil R-domain which, once bound to BtuB, places the colicin at an angle of ∼ 45° relative to the membrane plane (Kurisu et al, 2003; Sharma et al, 2007) This bound orientation is proposed to facilitate the search for their secondary receptor OmpF which is used as a translocator (Housden and Kleanthous, 2012; Jakes and Cramer, 2012). ColN, like ColIa, utilizes and binds to a single type of OMP, OmpF, as its receptor (Evans et al, 1996a) but its R-domain (ColN-R) has a very weak affinity for OmpF (Evans et al, 1996b)
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