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GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore.

ABSTRACT: We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-A and 28-A resolution, respectively. The structures were mushroom-shaped objects with a 125-A-diameter cap and a 100-A-long stem, consistent with earlier biochemical data. Thus, GroEL provides a platform for obtaining initial glimpses of a membrane protein structure in the absence of lipids or detergents and can function as a scaffold for higher-resolution structural analysis of the PA pore.

SUBMITTER: Katayama H

PROVIDER: S-EPMC2504863 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore.

Katayama Hiroo H Janowiak Blythe E BE Brzozowski Marek M Juryck Jordan J Falke Scott S Gogol Edward P EP Collier R John RJ Fisher Mark T MT

Nature structural & molecular biology 20080622 7

We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-A and 28- ...[more]

PMID: 18568038

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Onset of anthrax toxin pore formation.

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Solubilization and characterization of the anthrax toxin pore in detergent micelles.

Project description:Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self-associates to form a heptameric ring-shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore-like state. N-tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion-competent 440-kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore-like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14-solubilized pore showed an extended, mushroom-shaped structure. Circular dichroïsm measurements revealed an increase in alpha helix and a decrease in beta structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2beta2-2beta3 loop transforms into the transmembrane segment of the beta-barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to alpha structure is dependent on residues of the luminal 2beta11-2beta12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.

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A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore.

Project description:The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.

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Project description:We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN ) 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 LFN -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN -PA nanodisc structures and use of advanced automated particle selection methods.

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Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles.

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