Atomic structure of anthrax protective antigen pore elucidates toxin translocation

Abstract

Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells1. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet2,3,4,5,6,7,8,9. Although atomic structures of protective antigen prepores are available10,11,12,13,14, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.

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Figure 1: CryoEM reconstruction of the PA pore.
Figure 2: Atomic model of the PA pore.
Figure 3: Translocation channel of the PA pore.
Figure 4: Structure of the Φ-clamp.
Figure 5: Conversion from prepore to pore.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Three-dimensional cryoEM density maps of anthrax PA pore have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6224 (intact PA pore) and EMD-6225 (lacking the membrane-spanning β-barrel). The coordinates of atomic model of anthrax PA pore have been deposited in the Protein Data Bank under accession number 3J9C.

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health (AI094386/AI046420 and GM071940 to Z.H.Z., and AI022021 to R.J.C.), the American Heart Association (Postdoctoral Fellowship 14POST18870059 to J.J.), Damon Runyon Cancer Research Foundation Innovation Award (B.L.P.), and National Science Foundation (CAREER Award CHE-1351807 to B.L.P.). We thank the NERCE facility (the National Institutes of Health grant AI057159) for expression of toxin proteins. We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by University of California, Los Angeles, and by instrumentation grants from the National Institutes of Health (1S10RR23057, 1S10OD018111) and National Science Foundation (DBI-1338135). We thank L. Jin for initial efforts on this project and J. Feigon for discussion.

Author information

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Authors

Contributions

J.J. designed and performed the experiments, analysed data, and wrote the paper; Z.H.Z. initialized and supervised the research, analysed data, and wrote the paper. B.L.P. and R.J.C. analysed data and wrote the paper.

Corresponding author

Correspondence to Z. Hong Zhou.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Negative-stain and cryoEM of the PA pore.

a, Negative-stain EM micrograph of PA pore particles. Some representative top-view and side-view particles are selected with circles and squares, respectively. b, A full-size drift-corrected cryoEM micrograph of 3,710 pixels × 3,710 pixels of PA pore particles acquired from a Gatan K2 Summit direct electron detection camera, at 300 kV accelerating voltage, –2.7 µm defocus, and a total dose of 39 electrons per square ångström. Some representative side-view particles of PA pore are indicated by arrows. c, Power spectrum of the cryoEM micrograph in b. d, Representative cryoEM two-dimensional class averages of particles at different orientations. eh, Superimposition of representative regions of the cryoEM map (mesh) with the atomic model (stick), including α-helix (e), β-strand (f), loop (g), and Ca2+ ions (green spheres in h).

Extended Data Figure 2 Resolution estimation of the cryoEM single particle reconstruction of the PA pore.

a, ‘Gold standard’ FSC between two independently refined maps with an auto-mask that was corrected by phase randomization. The resolution was estimated by the ‘gold standard’ FSC at 0.143 criterion42. b, FSC of the final atomic model versus the final cryoEM map (black); of a model refined in the first of the two independent maps used for the ‘gold standard’ FSC versus the same map (red) and versus the second independent map (blue). c, Surface view and cut-through view of the unsharpened cryoEM map coloured by local resolution estimated by ResMap43. d, Euler angle distribution of all particles used for the final three-dimensional reconstruction.

Extended Data Figure 3 Fitting of domain 4 into the cryoEM map of PA pore.

a, The crystal structure of domain 4 (purple ribbons) from PA prepore (PDB accession number 1TZO) fits the cryoEM map (grey surface) of PA pore with a good agreement (cross-correlation coefficient: 0.91). b, Translocation of domain 4 during the prepore to pore conversion. Domain 4 is shifted inwards as a rigid-body for 4 Å from the prepore conformation (dark cyan) to the pore conformation (purple).

Extended Data Figure 4 Comparison of the structures of the PA pore, α-haemolysin (PDB accession number 7AHL), and Vibrio cholerae cytolysin (PDB accession number 3O44).

These three complexes are in their membrane-inserted forms. The approximate position of the lipid bilayer is illustrated with grey shade. The host cell receptor (TEM8 or CMG2) of PA that binds to domain 4 (grey ribbons) is schematically illustrated.

Extended Data Figure 5 Assembling of the Φ-clamp in the PA pore.

a, Comparison of the Φ-clamp in the PA pore with its corresponding region in the PA prepore. The residues Asp426 and Phe427 missing in the crystal structure of the PA63 prepore (PDB accession number 1TZO) are modelled based the crystal structure of the PA83 monomer (PDB accession number 1ACC). b, Close-up top view of the Φ-clamp region in the PA pore. The 2β7–2β8 and 2β10–2β11 loops are coloured in cyan and orange, respectively. The hydrogen bonds between Ser428, Asn399, and Lys397′, which form a chain tethering the 2β7–2β8 and 2β10–2β11 loops together, are depicted with dashed lines.

Extended Data Figure 6 Conformational changes of domain 2c between the PA prepore and the PA pore.

a, Superimposition of domains 2c of the PA pore and the crystal structure of the PA heptameric prepore (PDB accession number 1TZO). The dashed boxes highlight the conformational difference of the 2β10–2β11 loop between these two structures. Residues Asp426 and Phe427 are not solved in the crystal structure of PA prepore because of their flexibility. b, Superimposition of domains 2c of the PA pore and the PA octameric prepore (PDB accession number 3HVD). Note the similarity of the 2β10–2β11 loop (dashed box) between these two structures. c, Superimposition of domains 2c of the PA pore and the PA prepore (monomer; PDB accession number 3TEW). df, Close-up views of the rearrangements of the 2β10–2β11 loop (d), the 2β7–2β8 loop (e), and the 2β5–2β6 and 2β12–2β13 loops (f) during the conversion from prepore to pore. Val377 and Leu378 of the 2β5–2β6 loop and Val455 and Tyr456 of the 2β12–2β13 loop are flipped upside down to obtain the β-strand conformation in the PA pore, leading to an extension of 2β6 and 2β12 (f). CryoEM densities (mesh) corresponding to these loops are displayed to the right of df with atomic models superimposed, showing unambiguous atomic modelling. The superimpositions of the cryoEM map and the atomic model are shown in views different from the left panels for clarity. The conformational changes of these loops result in a more compact domain 2c with a decrease of 567 Å2 in its surface area.

Extended Data Figure 7 Compact assembly of the PA pore.

a, Surface views of two neighbouring protomers (the same domains in the two protomors are in different shades of the same colours) of the PA pore and the PA prepore (PDB accession number 1TZO) visualized from inside the PA heptamer. Domains 1′, 2c, 2s, and 3 are coloured differently and domain 4 is not shown. The inter-protomer interface in the PA prepore is largely formed by domains 1′ and 3, and domain 2 only contributes to this interface by its membrane insertion loop and carboxy (C)-terminal region (2β13, 2β14, and 2α3). In the PA pore, the convergence of domain 2c creates an inter-protomer interface without any gap, with an increase of interface area on domain 2c from 1,247 Å2 of the PA prepore to 2,106 Å2 of the PA pore as calculated using PISA49 (http://www.ebi.ac.uk/pdbe/pisa/). Additionally, formation of the β-barrel also leads to extensive contacts, creating a new interface area of 1,195 Å2 between two protomers. b, Schematic of the conversion of 2β2, 2β3, 2α1, and the membrane insertion loop of the PA prepore to 2β2s and 2β3s of the PA pore. Hydrogen bonds between 2β2 and 2β3, which are depicted with dashed lines, are maintained during the conversion. By contrast, 2α1 and the connecting loops have to be fully unfolded and converted into β-strands that collectively assemble the β-barrel of the PA pore. Although the detailed events of β-barrel formation are not yet clear, it is likely that assembly starts from the top in a zipper-like manner. A favourable scenario is that the convergence of domain 2c would place the top ends of the β-strands close to each other to form a short β-barrel, which could extend by pulling more residues together via formation of ordered hydrogen bonds until it reaches the bottom end. It is less favourable that the assembly starts from other regions because disordered hydrogen bonds and hydrophobic interactions could generate enormous non-productive, possibly irreversible pairings between strands.

Extended Data Figure 8 CryoEM single-particle reconstruction of a subset of particles lacking the 14-stranded β-barrel.

a, Surface views of the cryoEM map lacking the β-barrel. The map is unsharpened and has a resolution of 3.6 Å. b, Cross-section side view of superimposition of the unsharpened cryoEM map with the atomic model of the intact PA pore, showing the cryoEM map has the same conformation as the PA pore except for the absence of the 14-stranded β-barrel. c, ‘Gold standard’ FSC (with an auto-mask that was corrected by phase randomization) between two independently refined maps. d, Top view of the Φ-clamp region of the cryoEM map (mesh) lacking the β-barrel superimposed with the atomic model of the PA pore (ribbons), showing correct assembling of the Φ-clamp in the cryoEM map.

Extended Data Table 1 Data collection and structure refinement statistics

Supplementary information

Video 1: CryoEM map of the PA pore at 2.9 Å resolution.

The surface view of the cryoEM map is rendered at different thresholds, rotated and rocked to show the overall structure, and then zoomed in to show the structural details of the Φ-clamp. Last, superimposition of the cryoEM map with the atomic model is shown. Note that the cryoEM densities of the side chains of some acidic residues (Glu and Asp) are not visible at the chosen density threshold, but are visible at a lower threshold. These exposed acidic residues are probably more sensitive to electron radiation damage. Related to Fig. 1. (AVI 12484 kb)

Video 2: Rotation of domain 2c during the conversion from prepore to pore

The structure of the PA prepore (light green, PDB ID: 3TEW) is morphed into the conformation of the PA pore. Domains 1', 2c, and 3 of the pore are colored in pink, orange red, and blue, respectively, as in related Fig. 5c. Domains 2s and 4 are not shown. (AVI 1862 kb)

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Jiang, J., Pentelute, B., Collier, R. et al. Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521, 545–549 (2015). https://doi.org/10.1038/nature14247

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