A Common Interaction for the Entry of Colicin N and Filamentous Phage into Escherichia coli

Hecht, Oliver; Ridley, Helen; Lakey, Jeremy H.; Moore, Geoffrey R.

doi:10.1016/j.jmb.2009.03.035

Cited by 17 publications

(54 citation statements)

References 48 publications

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“…It is well established that unfolded or only partly folded proteins in their native states fold into an ordered structure on binding a partner molecule/protein (7,8). A well-studied example is the binding of colicins to disordered regions of their OM receptors as a key step in their translocation into the target cell (12,37). Therefore, the intrinsically disordered region in the C-terminal end of LptC (residues 185 to 191) might be reorganized and folded upon binding to LptA.…”

Section: Discussionmentioning

confidence: 99%

“…Chromatography was carried out at room temperature in 50 mM NaH 2 PO 4 (pH 8.0)-100 mM NaCl at a flow rate of 0.5 ml/min and monitored by the eluate absorbance at 280 nm. The calibration curve was constructed by using the following standards (0.5 mg/ml): transferrin (81,000 Da), chicken ovalbumin (43,000 Da), chymotrypsin (21,500 Da), bovine cytochrome c (12,200 Da), and aprotinin (6,500 Da) (Sigma Aldrich, St. Louis, MO). LptC was injected at a concentration of 1 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

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New Insights into the Lpt Machinery for Lipopolysaccharide Transport to the Cell Surface: LptA-LptC Interaction and LptA Stability as Sensors of a Properly Assembled Transenvelope Complex

et al. 2011

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Lipopolysaccharide (LPS) is a major glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The peculiar permeability barrier of the OM is due to the presence of LPS at the outer leaflet of this membrane that prevents many toxic compounds from entering the cell. In Escherichia coli LPS synthesized inside the cell is first translocated over the inner membrane (IM) by the essential MsbA flippase; then, seven essential Lpt proteins located in the IM (LptBCDF), in the periplasm (LptA), and in the OM (LptDE) are responsible for LPS transport across the periplasmic space and its assembly at the cell surface. The Lpt proteins constitute a transenvelope complex spanning IM and OM that appears to operate as a single device. We show here that in vivo LptA and LptC physically interact, forming a stable complex and, based on the analysis of loss-of-function mutations in LptC, we suggest that the C-terminal region of LptC is implicated in LptA binding. Moreover, we show that defects in Lpt components of either IM or OM result in LptA degradation; thus, LptA abundance in the cell appears to be a marker of properly bridged IM and OM. Collectively, our data support the recently proposed transenvelope model for LPS transport.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

New Insights into the Lpt Machinery for Lipopolysaccharide Transport to the Cell Surface: LptA-LptC Interaction and LptA Stability as Sensors of a Properly Assembled Transenvelope Complex

et al. 2011

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“…A substantial conformational change in phage g3p upon interaction with TolA was also observed . Previous NMR studies on colicin N and the N1 domain of the filamentous phage g3p protein demonstrated that TolA-III changes its conformation upon binding to colicin N but retains its global fold Deprez et al, 2002Deprez et al, , 2005Hecht et al, 2009). The nature of these structural changes would be extremely useful in elucidating the events of translocation, and therefore a construct of the T-domain TolA complex was constructed.…”

Section: Resultsmentioning

confidence: 99%

Bacterial toxin colicin N-T domain structure changes to ordered state upon binding C-terminal domain of TolA

Ulusu¹,

Şentürk²,

Gedikli³

et al. 2014

Turk J Biol

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IntroductionColicins are plasmid-encoded bactericidal proteins produced by immune Escherichia coli that are active against sensitive E. coli and its closely related cells. Their toxic activities are of various types: some colicins form ion channels in the cytoplasmic membrane of sensitive cells, while others act as nucleases that degrade DNA or 16S RNA in the cytoplasm. One colicin, colicin M, inhibits the biosynthesis of murein (Lakey et al., 1994). Their toxic activities against target cells are known to occur in 3 distinct stages (Figure 1). First is receptor recognition and binding, where colicins bind to a specific receptor at the cell surface. Second is the translocation step, where colicins cross both the outer membrane and the periplasm to reach their cellular target (Lakey et al., 1994;Lazdunski, 1995). The final stage is the killing action, where colicins exert their lethal effects by forming a pore in the cytoplasmic membrane (Lazdunski, 1995), by DNAse or RNAse activity, or by inhibiting murein biosynthesis in the cytoplasm (Lakey et al., 1994).Colicins have 3 linearly organized functional domains, each domain implicated in a specific stage of the colicin activity. As shown in Figure 1, the central domain (R-domain) is responsible for the receptor-binding activity. The N-terminal domain (T-domain) is involved in translocation. The C-terminal domain (P-domain) carries out the lethal activity; this domain either forms a voltagegated pore in the cytoplasmic membrane or digests nucleic acids in the cytoplasm (Raggett et al., 1998;Vetter et al., 1998;Papadokus et al., 2011).Colicin N is a group A (Tol-dependent) pore-forming colicin whose target receptor is the E. coli outer membrane protein OmpF (Pugsley, 1987). It is composed of a largely unstructured T-domain linked by a glycine-rich sequence to a central R-domain containing a 6-stranded β-sheet structure. The R-domain is connected to the P-domain (a 10 α-helical structure) by means of a 65 Å α-helix (El-Kouhen et al., 1993;Gokce et al., 2000). While receptor binding and pore formation have been extensively studied, much remains unknown about the translocation step. Colicin N translocation requires 3 members of the Tol locus: TolA, TolQ, and TolR (Webster, 1991). TolQ and TolR are integral membrane proteins of the E. coli inner membrane and there is no evidence that they reach across Abstract: Colicin N is a bacterial toxin that kills Escherichia coli and related cells. Its mode of action is of interest in protein import and toxicology. Colicin N translocates across the E. coli outer membrane and periplasm by interacting with several receptors. The translocation process involves the interaction of the colicin N with the outer membrane porin OmpF and subsequently with the integral membrane protein TolA. The N-terminal domain of colicin N is involved in the import process. TolA consists of 3 domains. The N-terminal domain of colicin N interacts with the C-terminal domain of TolA at later stages of the translocation process. Our aim was to produce a large ...

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“…Resonance assignments of 13 C/ 15 N labelled TolA‐III (0.6 mM) in complex with unlabelled ColA 53–107 (1 mM) were obtained from 1 H– 15 N HSQC, CBCA(CO)NH, HNCACB and HNCA spectra at 27 °C. NMR spectra were processed using NMRPipe [14] and analysed in NMRView [15] as described previously [10]. NH chemical shift variations were calculated using the formula: {(Δ δ H ) 2 + ( γ N / γ H ∗ Δ δ N ) 2 } 2 .…”

Section: Methodsmentioning

confidence: 99%

Characterisation of the interaction of colicin A with its co‐receptor TolA

Hecht

Zhang

et al. 2010

FEBS Letters

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Edited by Stuart Ferguson Keywords:Intrinsically disordered protein Colicin A TolA NMR Protein-protein interaction a b s t r a c t Colicin A enters Escherichia coli cells through interaction with endogenous TolA and TolB proteins. In vitro, binding of the colicin A translocation domain to TolA leads to unfolding of TolA. Through NMR studies of the colicin A translocation domain and polypeptides representing the individual TolA and TolB binding epitopes of colicin A we question if the unfolding of TolA induced by colicin A is likely to be physiologically relevant. The NMR data further reveals that the colicin A binding site on TolA is different from that for colicin N which explains why there is a difference in colicin toxicity for E. coli carrying a TolA-III homologue from Yersina enterocolitica in place of its own TolA-III.

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A Common Interaction for the Entry of Colicin N and Filamentous Phage into Escherichia coli

Cited by 17 publications

References 48 publications

New Insights into the Lpt Machinery for Lipopolysaccharide Transport to the Cell Surface: LptA-LptC Interaction and LptA Stability as Sensors of a Properly Assembled Transenvelope Complex

New Insights into the Lpt Machinery for Lipopolysaccharide Transport to the Cell Surface: LptA-LptC Interaction and LptA Stability as Sensors of a Properly Assembled Transenvelope Complex

Bacterial toxin colicin N-T domain structure changes to ordered state upon binding C-terminal domain of TolA

Characterisation of the interaction of colicin A with its co‐receptor TolA

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