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Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex

Nature Structural & Molecular Biology volume 27, pages 288–296 (2020)Cite this article

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Abstract

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.

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Fig. 1: Cryo-EM density maps of Ib pores.
Fig. 2: Atomic model of the Ib-pore.
Fig. 3: Atomic model of the Ia-bound Ib-pore.
Fig. 4: Unfolding of Ia N-terminus.
Fig. 5: Comparison of Ia-bound Ib and LF-bound PA.
Fig. 6: Translocation model of Ia via Ib-pore.

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Data availability

Cryo-EM maps and coordinates were deposited to the Electron Microscopy Data Bank and Protein Data Bank with the accession codes EMDB-0721 and PDB 6KLX for the Ib-pore, EMDB-0713 and PDB 6KLO for the Ia-bound Ib-pore with the short stem, and EMDB-0720 and PDB 6KLW for the Ia-bound Ib-pore with the long stem, respectively.

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Acknowledgements

We thank H. Murata for the initial purification of Ib and JI. Kishikawa for help in cryo-EM analysis. H.T. thanks M. Nagahama and M. Oda for helpful comments on the studies. This work was supported by JSPS KAKENHI grant numbers 18K06170 and 17K15095. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) under grant number JP19am0101072 (support number 1232). This work was partially supported by grants-in-aid from ‘Nanotechnology Platform’ of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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  1. These authors contributed equally: Tomohito Yamada, Toru Yoshida, Akihiro Kawamoto.

Authors and Affiliations

  1. Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto, Japan

    Tomohito Yamada, Toru Yoshida & Hideaki Tsuge

  2. Institute for Protein Dynamics, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto, Japan

    Toru Yoshida & Hideaki Tsuge

  3. Institute for Protein Research, Osaka University, Suita, Osaka, Japan

    Akihiro Kawamoto & Kenji Iwasaki

  4. Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Ibaraki, Osaka, Japan

    Kaoru Mitsuoka

  5. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan

    Kenji Iwasaki

  6. Center for Molecular Research in Infectious Diseases, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto, Japan

    Hideaki Tsuge

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Contributions

T. Yamada, T. Yoshida, A.K. and H.T. participated in the research design and data analyses; T. Yamada prepared the Ib-pore and Ia-bound Ib-pore for cryo-EM; T. Yamada, A.K., K.M. and K.I. performed cryo-EM data acquisition and image processing; T. Yoshida performed atomic-model building, structure refinement, and analyses; all authors contributed to writing the manuscript and H.T. supervised the project.

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Correspondence to Hideaki Tsuge.

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Extended data

Extended Data Fig. 1 Sample preparation of iota toxin.

a, SDS-PAGE (samples are not heat-denatured) showing ethanol-induced Ib oligomerization. After treatment with α-chymotrypsin, Ib monomer was oligomerized by ethanol addition. Oligomerization efficiency reached ~100% in the presence of 10% ethanol. High concentration of ethanol (~20%) caused aggregation, as reflected by the band above the Ib-oligomer. b, Sample preparation for the first data set: top, Density gradient ultracentrifugation. bottom, SDS-PAGE of fractions 21–24, showing that these fractions included the Ib oligomer along with small amounts of Ia. c, Sample preparation for the 2nd data set: top, Density gradient ultracentrifugation of Ib-pore without Ia. bottom, SDS-PAGE of fractions 17–20. Ia was added at three-fold molar excess prior to 2nd data set collection.

Extended Data Fig. 2 Single particle analysis of 1st data (Ib-pore and low resolution Ia-bound Ib-pore).

a, Representative microscope images at pH 5.5 and pH 7.5. Images for analysis were taken only at pH 7.5, due to aggregation at pH 5.5. Scale bar: 100 nm. b–e, Ib-pore analysis. f, g, Ia-bound Ib-pore analysis. These analyses were performed individually from the same image set (1st data). b, Flow chart of cryo-EM image processing for Ib-pore. c, Final 3D reconstruction map color-coded according to local resolution. d, Angular distribution of particles projected to the map. The angles of the projected particle are shown as 1/7 of a sphere because this analysis was performed with C7 symmetry. e, Gold-standard Fourier shell correlation (FSC) curve of final map and FSC curve for cross-validation between the map and model. f, Flow chart of cryo-EM image processing for Ia-bound Ib-pore. After initial 3D refinement, density around the Ib pore stem was subtracted. g, Final 3D reconstruction map. h, Angular distribution of particles projected to the map.

Extended Data Fig. 3 Single particle analysis of the 2nd data (high resolution Ia-bound Ib-pore).

a, Representative microscope image of the 2nd sample at pH 7.5. Scale bar: 100 nm. b, Flow chart of cryo-EM image processing. c, Final 3D reconstruction map of Ia-bound Ib-pore with a short stem, color-coded according to local resolution. Ia is clearly observed. d, Angular distribution of particles projected to the map shown in c. e, Gold-standard FSC curve of the final map shown in c and FSC curve for cross-validation between the map and model. f, Final 3D reconstruction map of Ia-bound Ib-pore with a long stem, color-coded according to local resolution. Ia is unclearer compared to map c. g, Angular distribution of particles projected to the map shown in f. h, Gold-standard FSC curve of the final map shown in f and FSC curve for cross-validation between the map and model.

Extended Data Fig. 4 Cryo-EM density maps and models.

Representative cryo-EM density maps and models.

Extended Data Fig. 5 Amino acid sequence alignment of domain 1′, 2, and 3.

The secondary structures of Ib-pore and PA-pore (PDB ID: 3J9C) are shown above and below the sequence, respectively. The amino acids numbers including the signal sequences are shown in Ib, CSTb, CDTb, and C2II. The amino acids numbers without the signal sequence are shown in PA. Uniprot IDs: Ib, Q46221; CSTb, o06498; CDTb, o32739; C2II, o86171; PA, P13423.

Extended Data Fig. 6 Comparison between Ib and PA.

a, Ca-binding sites. Cryo-EM density map are overlaid in Ib. The calcium ions are shown as green spheres. Lowercase “m” in parentheses indicates that the main chain forms a coordination bond. b, Domain IV. Cryo-EM density maps and models are shown. PA PDB ID: 3J9C. Extra maps correspond to domain IV. The C-terminus of the Ib domain IV is invisible. c, Amino acid residues comprising inner surfaces of pores. left, A protomer on Ib surface model is colour coded according to the properties of amino acids. right, Schematic representations of amino acid residues consisting of stem regions. d, Cut-away surface electrostatic potential at pH 7.0 and 5.5.

Extended Data Fig. 7 Iota toxin intoxication strategy.

(1) Pro-Ib monomer binds to LSR and is activated by proteolysis, which triggers oligomerization to form the prepore and also the Ib-pore (short) as a metastable state (2) Ia binds to the Ib-prepore and Ib-pore (short) at pH 7.5. (3) Endosome acidic pH triggers conversion of these states to Ib-pore (long) or Ia-bound Ib-pore (long) at pH 5.5.

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Video

Unfolding of the Ia N-terminal caused by binding to Ib-pore. A constriction site, the NSQ-loop, changes the conformation when Ia binds to Ib-pore and makes the Ia N-terminal α-helix unfold and tilt.

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Yamada, T., Yoshida, T., Kawamoto, A. et al. Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex. Nat Struct Mol Biol 27, 288–296 (2020). https://doi.org/10.1038/s41594-020-0388-6

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