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The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein.

Johnson CL, Ridley H, Marchetti R, Silipo A, Griffin DC, Crawford L, Bonev B, Molinaro A, Lakey JH - Mol. Microbiol. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK.

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SPR reveals ColN‐R is responsible for LPS binding. Histidine‐tagged ColN domain combinations (500 nM) were injected for 60 s at a flow rate of 5 μl min−1 over a Ni2+ charged NTA chip at 0 s followed as indicated by black arrows by either (A) 100 μM Rc LPS, (B) 100 μM Rd LPS for 60 s or (C) ColN(V94C/Q179C) in either an oxidized (ColNOX) or a reduced (ColNRED) state was used. Large drops in signal after ColN construct injections are due to buffer effects. The subsequent differences the quantity of bound material reflected in the different RU values after the binding of different ColN constructs largely correlate with the different masses of the constructs, e.g. ColN 42, ColN‐P 21.5, ColN‐TR 20.5, ColN‐R 11.5 and ColN‐T 9 kDa. The relatively high value for ColN‐T may reflect the denser packing possibly with a natively disordered domain.
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mmi12568-fig-0003: SPR reveals ColN‐R is responsible for LPS binding. Histidine‐tagged ColN domain combinations (500 nM) were injected for 60 s at a flow rate of 5 μl min−1 over a Ni2+ charged NTA chip at 0 s followed as indicated by black arrows by either (A) 100 μM Rc LPS, (B) 100 μM Rd LPS for 60 s or (C) ColN(V94C/Q179C) in either an oxidized (ColNOX) or a reduced (ColNRED) state was used. Large drops in signal after ColN construct injections are due to buffer effects. The subsequent differences the quantity of bound material reflected in the different RU values after the binding of different ColN constructs largely correlate with the different masses of the constructs, e.g. ColN 42, ColN‐P 21.5, ColN‐TR 20.5, ColN‐R 11.5 and ColN‐T 9 kDa. The relatively high value for ColN‐T may reflect the denser packing possibly with a natively disordered domain.

Mentions: SPR was used to identify which regions of ColN were responsible for LPS binding. Various C‐terminally histidine‐tagged ColN domain mutants were injected at equal molar concentrations over a Ni2+ charged NTA surface followed by either Rc LPS (Fig. 3A) or Rd LPS (Fig. 3B). The data demonstrate that all R‐domain containing constructs (i.e. ColN, ColN‐TR and ColN‐R) bind to Rc LPS while ColN‐T and ColN‐P do not (Fig. 3A). LPS dissociates more slowly from ColN or ColN‐R than from ColN‐TR which nevertheless binds significant amounts of LPS. Thus ColN‐R serves as the minimal Rc LPS binding unit and it binds with a similar kinetic profile in both the isolated construct and the full‐length ColN protein. In agreement with the genetic screen (Sharma et al., 2009) none of the constructs tested bound to Rd LPS (Fig. 3B). All the colicin constructs possess a high net positive charge (pI = ∼ 9.0) and show weak, non‐specific, electrostatic binding to any negatively charged LPS which is revealed by small changes in response units (RUs) with Rd LPS (Fig. 3B) and for ColN‐T and ColN‐P with Rc LPS.


The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein.

Johnson CL, Ridley H, Marchetti R, Silipo A, Griffin DC, Crawford L, Bonev B, Molinaro A, Lakey JH - Mol. Microbiol. (2014)

SPR reveals ColN‐R is responsible for LPS binding. Histidine‐tagged ColN domain combinations (500 nM) were injected for 60 s at a flow rate of 5 μl min−1 over a Ni2+ charged NTA chip at 0 s followed as indicated by black arrows by either (A) 100 μM Rc LPS, (B) 100 μM Rd LPS for 60 s or (C) ColN(V94C/Q179C) in either an oxidized (ColNOX) or a reduced (ColNRED) state was used. Large drops in signal after ColN construct injections are due to buffer effects. The subsequent differences the quantity of bound material reflected in the different RU values after the binding of different ColN constructs largely correlate with the different masses of the constructs, e.g. ColN 42, ColN‐P 21.5, ColN‐TR 20.5, ColN‐R 11.5 and ColN‐T 9 kDa. The relatively high value for ColN‐T may reflect the denser packing possibly with a natively disordered domain.
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4114557&req=5

mmi12568-fig-0003: SPR reveals ColN‐R is responsible for LPS binding. Histidine‐tagged ColN domain combinations (500 nM) were injected for 60 s at a flow rate of 5 μl min−1 over a Ni2+ charged NTA chip at 0 s followed as indicated by black arrows by either (A) 100 μM Rc LPS, (B) 100 μM Rd LPS for 60 s or (C) ColN(V94C/Q179C) in either an oxidized (ColNOX) or a reduced (ColNRED) state was used. Large drops in signal after ColN construct injections are due to buffer effects. The subsequent differences the quantity of bound material reflected in the different RU values after the binding of different ColN constructs largely correlate with the different masses of the constructs, e.g. ColN 42, ColN‐P 21.5, ColN‐TR 20.5, ColN‐R 11.5 and ColN‐T 9 kDa. The relatively high value for ColN‐T may reflect the denser packing possibly with a natively disordered domain.
Mentions: SPR was used to identify which regions of ColN were responsible for LPS binding. Various C‐terminally histidine‐tagged ColN domain mutants were injected at equal molar concentrations over a Ni2+ charged NTA surface followed by either Rc LPS (Fig. 3A) or Rd LPS (Fig. 3B). The data demonstrate that all R‐domain containing constructs (i.e. ColN, ColN‐TR and ColN‐R) bind to Rc LPS while ColN‐T and ColN‐P do not (Fig. 3A). LPS dissociates more slowly from ColN or ColN‐R than from ColN‐TR which nevertheless binds significant amounts of LPS. Thus ColN‐R serves as the minimal Rc LPS binding unit and it binds with a similar kinetic profile in both the isolated construct and the full‐length ColN protein. In agreement with the genetic screen (Sharma et al., 2009) none of the constructs tested bound to Rd LPS (Fig. 3B). All the colicin constructs possess a high net positive charge (pI = ∼ 9.0) and show weak, non‐specific, electrostatic binding to any negatively charged LPS which is revealed by small changes in response units (RUs) with Rd LPS (Fig. 3B) and for ColN‐T and ColN‐P with Rc LPS.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK.

Show MeSH
Related in: MedlinePlus