A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

Farrance, Oliver E.; Hann, Eleanore; Kaminska, Renata; Housden, Nicholas G.; Kleanthous, Colin; Radford, Sheena E.; Brockwell, David J.

doi:10.1371/journal.pbio.1001489

Cited by 26 publications

(37 citation statements)

References 60 publications

(95 reference statements)

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“…The presence of the TolC box sequence serves to weaken interhelical interactions within the T domain and could facilitate its conversion to a more extended conformation that could then enter TolC as described above. For the DNase colicin E9, it has been demonstrated that the application of very low forces (Ͻ20 pN) is sufficient to trigger the dissociation of its immunity protein, which has a K d (dissociation constant) in the femtomolar range (41). It can be postulated that some comparably weak interaction between colicin E1 and TolC triggers its release from its BtuB receptor and an unfolding that would allow binding to and passage through TolC so that the channel-forming domain reaches its ultimate target, the bacterial inner membrane.…”

Section: Discussionmentioning

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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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: Discussionmentioning

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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“…DNase-specific Im proteins are small acidic inhibitors that bind to an exosite and inhibit colicin or pyocin activity indirectly by blocking substrate-binding [15]. Im proteins are known to dissociate from colicins at the cell surface in a pmf-dependent step by a process that is thought to involve structural remodeling of the nuclease [16,17]. …”

Section: Introductionmentioning

confidence: 99%

Structural and biophysical analysis of nuclease protein antibiotics

Klein

Wojdyla

Joshi

et al. 2016

Biochemical Journal

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Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity Im protein, Im9. In contrast with Im3, which when bound to the ribonuclease domain of the homologous colicin ColE3 makes contact with the translocation (T) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small-angle X-ray scattering data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules, even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.

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“…Single-molecule force spectroscopy is a commonly used tool for the mechanical characterization of polymers and biological molecules, such as proteins and DNA [1][2][3][4][5][6][7][8][9][10], and ligand-protein interactions [11][12][13][14][15][16][17][18][19]. These experiments can be performed using a variety of experimental setups, including optical tweezers [3,20], the atomic force microscope (AFM) [1,4], magnetic tweezers [21,22], and the biomembrane force probe [23].…”

Section: Introductionmentioning

confidence: 99%

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

et al. 2015

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Single-molecule force spectroscopy using an atomic force microscope (AFM) can be used to measure the average unfolding force of proteins in a constant velocity experiment. In combination with Monte Carlo simulations and through the application of the Zhurkov-Bell model, information about the parameters describing the underlying unfolding energy landscape of the protein can be obtained. Using this approach, we have completed protein unfolding experiments on the polyprotein (I 27) 5 over a range of pulling velocities. In agreement with previous work, we find that the observed number of protein unfolding events observed in each approach-retract cycle varies between one and five, due to the nature of the interactions between the polyprotein, the AFM tip, and the substrate, and there is an unequal unfolding probability distribution. We have developed a Monte Carlo simulation that incorporates the impact of this unequal unfolding probability distribution on the median unfolding force and the calculation of the protein unfolding energy landscape parameters. These results show that while there is a significant, unequal unfolding probability distribution, the unfolding energy landscape parameters obtained from use of the Zhurkov-Bell model are not greatly affected. This result is important because it demonstrates that the minimum acceptance criteria typically used in force extension experiments are justified and do not skew the calculation of the unfolding energy landscape parameters. We further validate this approach by determining the error in the energy landscape parameters for two extreme cases, and we provide suggestions for methods that can be employed to increase the level of accuracy in single-molecule experiments using polyproteins.

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A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

Abstract: A single-molecule force study shows that rapid dissociation of a high-affinity protein interaction can be triggered by site-specific remodelling of one protein partner, and that prevention of remodelling maintains avidity.

Cited by 26 publications

References 60 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

Structural and biophysical analysis of nuclease protein antibiotics

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

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