Cys−Cys Cross-Linking Shows Contact between the N-Terminus of Lethal Factor and Phe427 of the Anthrax Toxin Pore

Janowiak, Blythe E.; Jennings-Antipov, Laura D.; Collier, R. John

doi:10.1021/bi1017446

Cited by 9 publications

(7 citation statements)

References 23 publications

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“…3(B)] compared to the unliganded PA in the previous publication 6. This difference is consistent with the insertion of the unstructured amino terminus of the LF N into the translocon channel, as predicted by biochemical and biophysical studies 7, 10…”

Section: Resultssupporting

confidence: 85%

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

et al. 2013

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We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LF N ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (~43%). Three-dimensional analysis of negatively stained single particles revealed the LF N -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LF N -PA nanodisc structures and use of advanced automated particle selection methods.

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Section: Resultssupporting

confidence: 85%

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

et al. 2013

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“…Observing these different states of the engagement complex (pH 5.0 vs. pH 7.5, 1 LF N vs. 3 LF N ) would be useful in determining the position of the Phe clamp loop region and potentially defining unstructured regions of the LF N that may become structured upon binding to the pore prior to translocation at pH 5.0. There are existing crosslinking studies by the Collier group indicating this interaction is present at pH 5.0 [ 13 ]. Thus, there is precedence for this interaction and those cryo-EM structure collection experiments at pH 5.0 are currently underway.…”

Section: Discussionmentioning

confidence: 99%

“…Translocation of α-helical regions of LF are aided by the PA α-clamp [ 8 , 9 ]. LF translocation is gated by a ring of seven phenylalanine residues, termed the Phe clamp, located further down the PA pore lumen [ 10 , 11 , 12 , 13 ]. The directional translocation of LF depends on protonation of acidic residues, the electrostatic character of the PA pore lumen, and any residual positive charges on LF [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

“…The first step of this pore forming mechanism is based on the increased flexibility of the 2β 10 –2β 11 loop in various prepore crystal structures. Early characterization of LF-PA interactions showed the N-terminal tail of LF interacts with the Phe clamp of the PA pore at pH 5.0, which has since been verified using cysteine cross-linking [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

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Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain

Machen

Akkaladevi

Trecazzi

et al. 2017

Toxins

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The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.

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“…Prior to assembly, these cysteine PA pores, these constructs were shown to be functionally competent to transport K + ions across a potential gradient. To avoid multiple thiol attachments onto the thio-sepharose bead supports which in turn may inhibit effective release, hetero-heptameric complexes were formed using procedures developed by Janowiak et al 2009, 2011. Addition of pre-nanodisc cholate–POPC–MSP micelles surrounded the exposed hydrophobic tips and upon dialysis with biobeads (Bio-RAD), the pre-nanodiscs collapsed to nanodisc structures.…”

Section: Construction Of Purified Pa Pore Nanodiscs Alone Verified Bymentioning

confidence: 99%

Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

et al. 2015

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Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding ↔ refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors.

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Cys−Cys Cross-Linking Shows Contact between the N-Terminus of Lethal Factor and Phe427 of the Anthrax Toxin Pore

Cited by 9 publications

References 23 publications

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain

Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

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