Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

Abstract

We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LF N ) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF N , though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF N extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.

Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K + channel: The power of native nanodiscs

“…The use of amphipols to stabilize MPs has recently led to a high-resolution de novo structure determination of a cation channel at a resolution of 3.4 Å (41), breaking the sidechain resolution barrier. Together with promising studies using scaffold-protein nanodiscs (42,43), as well as initial studies with native nanodiscs (44), these new developments suggest that native nanodiscs may become a powerful alternative to enable high-resolution structural characterization of MPs in their native environment.…”

Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

“…This is likewise important for electron microscopy studies where affinity is more important than for crystallization chaperones since the concentrations are considerably lower. Additionally, the technology to collect EM data while the protein is still embedded in the nanodisc is being further developed and offers the opportunity to quickly provide microscopists ready made systems with customized fiducial markers (Frauenfeld et al, 2011; Wu et al, 2012; Choi et al, 2013; Gogol et al, 2013). …”

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

“…In order to construct PA pore structures that could be inserted into lipid nanodiscs while positioned on an immobilized support, three potential cysteine attachment sites were engineered into the anthrax PA prepore cap alone. Of these mutants, two cysteine PA heptamer variants (S186C and S206C) were successfully immobilized using thio-sepharose beads and these immobilized PA pores were transitioned at pH 7.5, 1 M urea as shown previously (Katayama et al 2010; Akkaladevi et al 2013; Gogol et al 2013). Prior to assembly, these cysteine PA pores, these constructs were shown to be functionally competent to transport K + ions across a potential gradient.…”

“…Thus, the lethal doses of the combined anthrax toxin are small. Specifically, we successfully constructed the assembled pre-endosomal complex (green arrows) using the cysteine mutant N-terminal portion of the anthrax lethal factor, previously developed to obtain structures for EM structure analysis (Akkaladevi et al 2013; Gogol et al 2013). Using a similar covalent attachment protocol, the N-terminal lethal factor (LF N ) E126C was linked to the biosensor tip through a mixed disulfide to orient the LF binding site on the BLI biosensor surface so that the next components of the anthrax toxin can be assembled in a stepwise manner (Fig.…”

“…6). Curiously, earlier cryo-EM imaging of the anthrax pore complex on quantifoil grids containing a thin carbon layer over the holes showed a preferential end-on view of the nanodiscs (Gogol et al 2013). Many anthrax pore nanodisc complexes were observed but the contrast of the protein was diminished due to the background carbon layer.…”