Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

Hands, Sarah; Holland, Lisa E.; Vankemmelbeke, Mireille; Fraser, L.J.; Macdonald, Colin; Moore, Geoffrey R.; James, Richard; Penfold, Christopher N.

doi:10.1128/jb.187.19.6733-6741.2005

Cited by 26 publications

(42 citation statements)

References 47 publications

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“…The results of previous ColE9 T-domain mutagenesis data can now be rationalized in the context of the TolB-ColE9 T pep32-47 complex structure (21)(22)(23). Mutations fall into two categories, those that abolish binding and those that result in weak binding to TolB.…”

Section: Resultsmentioning

confidence: 99%

“…A ''TolB box'' ( 35 DGSGW 39 ) has previously been described that is part of the natively unfolded T-domain where mutations (of non-glycine residues) abolish colicin toxicity (21,22). More recently, the potential TolB binding site has been extended by mutagenesis and NMR experiments (17,23), but it is still not clear whether the site is a contiguous or discontiguous epitope or whether regions outside the natively disordered domain are involved. Two complementary approaches were taken to delineate precisely the binding site in ColE9.…”

Section: Resultsmentioning

confidence: 99%

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Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

Loftus

Walker

Mate

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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155

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The natively disordered N-terminal 83-aa translocation (T) domain of E group nuclease colicins recruits OmpF to a colicin-receptor complex in the outer membrane (OM) as well as TolB in the periplasm of Escherichia coli, the latter triggering translocation of the toxin across the OM. We have identified the 16-residue TolB binding epitope in the natively disordered T-domain of the nuclease colicin E9 (ColE9) and solved the crystal structure of the complex. ColE9 folds into a distorted hairpin within a canyon of the six-bladed ␤-propeller of TolB, using two tryptophans to bolt the toxin to the canyon floor and numerous intramolecular hydrogen bonds to stabilize the bound conformation. This mode of binding enables colicin side chains to hydrogen-bond TolB residues in and around the channel that runs through the ␤-propeller and that constitutes the binding site of peptidoglycan-associated lipoprotein (Pal). Pal is a globular binding partner of TolB, and their association is known to be important for OM integrity. The structure is therefore consistent with translocation models wherein the colicin disrupts the TolB-Pal complex causing local instability of the OM as a prelude to toxin import. Intriguingly, Ca 2؉ ions, which bind within the ␤-propeller channel and switch the surface electrostatics from negative to positive, are needed for the negatively charged T-domain to bind TolB with an affinity equivalent to that of Pal and competitively displace it. Our study demonstrates that natively disordered proteins can compete with globular proteins for binding to folded scaffolds but that this can require cofactors such as metal ions to offset unfavorable interactions.native disorder ͉ thermodynamics ͉ toxin

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

Loftus

Walker

Mate

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

155

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“…The N-terminal domains of co-licins have been postulated to be poorly structured regions based on the low amount of ordered secondary structures observed by circular dichroism, a weak dispersion of NMR signals, the absence of detection of corresponding electron density during X-ray crystallography studies, and high susceptibility to proteolytic degradation (6,125,132,147,496,546,607,650). Biophysical studies have demonstrated that the TolB binding sequence of colicin E9 becomes more structured when in interaction with TolB (125,248,431,632). Similarly, transition of the N-terminal domain of colicin N from a disordered state to a folded conformation is induced upon TolA C-terminal binding (5,6).…”

Section: Tol-dependent Colicinsmentioning

confidence: 99%

“…Overall, these data suggest that the N-terminal segments of the group A colicin translocation domains are disordered in their native states and fold into an ordered structure when binding a Tol subunit. This recognition mechanism has been suggested to contribute to the specificity of the recognition event, to allow a protein to bind several target partners, and to provide large protein binding surfaces in relatively small regions (168,241,248,362). Hierarchy of contacts during transit.…”

Section: Tol-dependent Colicinsmentioning

confidence: 99%

Colicin Biology

Cascales

Buchanan

Duché

et al. 2007

Microbiol Mol Biol Rev

984

1,324

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SUMMARY Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.

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“…However, the details of colicin entry through OmpF are largely unknown. The cytotoxicity of colicin E3 is dependent on the interaction of the periplasmic TolB protein with the TolB box spanning residues 34 to 46 (18,19), which is contained in the 83-residue N-terminal translocation subdomain-the T83 segment. It is likely that the high Gly content of this subdomain and its resulting flexibility (11,41) allow it to traverse the outer membrane through a translocation complex that contains OmpF (25,47) and to interact with the TolB protein in the periplasmic space (6).…”

mentioning

confidence: 99%

Minimum Length Requirement of the Flexible N-Terminal Translocation Subdomain of Colicin E3

Sharma

2007

J Bacteriol

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The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.Transport of proteins across membranes in import and secretion is known to require an assembly of integral membrane proteins that provides passage across the hydrophobic membrane (29,36). Such an assembly is utilized by colicins, which parasitize receptors in the outer membrane to gain entry into the cell (8,27,28,46). Colicins are highly specific in that they kill only those cells that (i) contain the receptor protein to which colicin binds and (ii) do not produce the cognate immunity protein, providing a selective advantage to the colicinproducing cell in a competitive environment (37).Colicin production is under SOS control, forming part of the stress response of the cell (20,43). Colicins are produced in complex with a small (molecular mass of ϳ10 kDa) immunity protein that protects the cell from both exogenous and endogenous colicins (7,22). Colicins have been divided into two groups based on the protein translocation network utilized to cross the outer membrane. Group A colicins, such as the E colicins, utilize the Tol system, consisting of TolA, TolB, TolQ, TolR, and Pal proteins (15,28,31). Group B colicins, such as colicins B, D, Ia, and Ib, utilize the Ton system consisting of TonB, ExbB, and ExbD proteins (8, 16). Colicin E3, a 16S rRNase (5), possesses a domain structure (34,35,41) as found in other colicins (1,2,13,14,45). It consists of three separate domains that have distinct roles. Its 315-residue N-terminal T domain functions in cell translocation and entry (1, 2, 41). The central 135-residue receptor (R) domain binds to BtuB (25), and the C-terminal domain contains the catalytic activity (34,35,41). The first step of E3 entry into the cell involves binding of the R domain to BtuB (25), a 22-stranded ␤-barrel outer membrane receptor whose metabolic function is transport of cyanocobalamin (vitamin B 12 ) acro...

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Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

Cited by 26 publications

References 47 publications

Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

Colicin Biology

Minimum Length Requirement of the Flexible N-Terminal Translocation Subdomain of Colicin E3

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