Colicin Occlusion of OmpF and TolC Channels: Outer Membrane Translocons for Colicin Import

Zakharov, Stanisłav D.; Eroukova, Veronika; Rokitskaya, Tatyana I.; Zhalnina, Mariya V.; Sharma, Onkar; Loll, Patrick J.; Zgurskaya, Helen I.; Antonenko, Yuri N.; Cramer, William A.

doi:10.1529/biophysj.104.046151

Cited by 78 publications

(134 citation statements)

References 70 publications

(105 reference statements)

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“…Just as the T domains of enzymatic colicins have been shown to bind to and enter their OmpF translocator, the T domain of colicin E1 occludes TolC channels in planar lipid bilayer membranes (17). Binding of some somewhat-shorter E1 T domain peptides was also detected by similar occlusion of TolC channels in planar bilayers and by coelution of those peptides with TolC on a sizing column (28).…”

Section: Importancementioning

confidence: 99%

“…1B) facilitates a search in two dimensions, via lateral diffusion and a "fishing pole" mechanism, by which the highly unstructured N-terminal T domain finds a copy of its OmpF translocator (8,13). For colicins E3 and E9, segments of their T domains were shown to be bound inside the pore of OmpF (14)(15)(16), and their T domains have also been shown to occlude OmpF channels in planar lipid bilayer membranes (16,17). For colicin Ia, a pore-forming colicin, a second copy of its Cir receptor serves as its translocator (10,18).…”

Section: Importancementioning

confidence: 99%

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The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Jakes

2017

J Bacteriol

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Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCEThe Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.KEYWORDS TolC, colicins, drug efflux, membrane translocation, toxins E scherichia coli competes for scarce resources by making plasmid-encoded protein toxins called colicins, which efficiently kill closely related bacteria. All of the few dozen or so colicins that have been identified kill their targets by one of a few basic mechanisms: (i) making an ion-permeable channel in the inner membrane of the target cell, which depolarizes and kills the cell (1); (ii) enzymatically cleaving its rRNA, tRNA, or DNA in the cytoplasm (2, 3); or (iii) degrading peptidoglycan precursors in the periplasm (4, 5). Regardless of their ultimate killing mechanism, all colicins must cross at least the outer membrane in order to reach their targets. The way one particular colicin, E1, makes that transit is the subject of this study.Colicins have a well-defined domain structure, with the killing (catalytic or channelforming) domain at the ...

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Section: Importancementioning

confidence: 99%

Section: Importancementioning

confidence: 99%

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Jakes

2017

J Bacteriol

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“…Although the mechanisms of translocation have not yet been definitively established for any colicin of either group, three possibilities have been proposed. The colicin might translocate outside the receptor, at the lipid-protein interface (Bainbridge et al, 1998); it might bind to one OM protein as receptor and use a second as translocation channel (Zakharov et al, 2004); or it might translocate through a single channel (Hilsenbeck et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Studies on colicin B translocation: FepA is gated by TonB

Devanathan

Postle

2007

Molecular Microbiology

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SummaryColicin B is a 55 kDa dumbbell-shaped protein toxin that uses the TonB system (outer membrane transporter, FepA, and three cytoplasmic membrane proteins TonB/ExbB/ExbD) to enter and kill Escherichia coli. FepA is a 22-stranded b-barrel with its lumen filled by an amino-terminal globular domain containing an N-terminal semiconserved region, known as the TonB box, to which TonB binds. To investigate the mechanism of colicin B translocation across the outer membrane, we engineered cysteine (Cys) substitutions in the globular domain of FepA. Colicin B caused increased exposure to biotin maleimide labelling of all Cys substitutions, but to different degrees, with TonB as well as the FepA TonB box required for all increases. Because of the large increases in exposure for Cys residues from T13 to T51, we conclude that colicin B is translocated through the lumen of FepA, rather than along the lipid-barrel interface or through another protein. Part of the FepA globular domain (residues V91-V142) proved relatively refractory to labelling, indicating either that the relevant Cys residues were sequestered by an unknown protein or that a significant portion of the FepA globular domain remained inside the barrel, requiring concomitant conformational rearrangement of colicin B during its translocation. Unexpectedly, TonB was also required for colicininduced exposure of the FepA TonB box, suggesting that TonB binds FepA at a different site prior to interaction with the TonB box.

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“…It is thought that the T-domain is then lowered into the periplasm through the lumen of OmpF from where it must locate the 45-kDa translocation portal TolB to trigger cell entry (19)(20)(21). In the present study, we set out to determine the molecular basis for TolB recruitment by ColE9 in the periplasm.…”

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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.

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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Colicin Occlusion of OmpF and TolC Channels: Outer Membrane Translocons for Colicin Import

Cited by 78 publications

References 70 publications

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Studies on colicin B translocation: FepA is gated by TonB

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

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