Research ArticleMICROBIOLOGY

Common architecture of Tc toxins from human and insect pathogenic bacteria

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Science Advances  16 Oct 2019:
Vol. 5, no. 10, eaax6497
DOI: 10.1126/sciadv.aax6497
  • Fig. 1 Structures of five TcAs.

    (A to E) Cryo-EM density maps of Pl-TcdA1, Pl-TcdA4, Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB, respectively, with the average resolutions according to 0.143 Fourier shell correlation. The color gradient from light to dark represents the pore domain with the TcB-binding domain, the α-helical shell, the β sheet domains, and the linker. (F to J) Structures of the TcA protomers. Pl-TcdA4 does not contain RBD A. RBD C was not well resolved in Xn-XptA1 and Mm-TcdA4 and is therefore not included in the models. Yp-TcaATcaB does not contain any RBD. The unique coiled-coil domain of Yp-TcaATcaB is highlighted in dark blue. Ninety-nine residues (amino acids 1140 to 1239) of the neuraminidase-like domain and the first 57 residues at the N-terminus (dotted line) Yp-TcaATcaB could not be built. The densities of the domains that could not be built are shown to indicate their location (H to K). The N-terminus Yp-TcaATcaB (residues 1 to 57) is depicted as red dotted line (J). (K) Domain organization of Pl-TcdA1, Pl-TcdA4, Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB. 1 = helical shell, 2 = RBD A, 3 = neuraminidase-like domain, 4 = RBD B, 5 = RBD C, 6 = RBD D, 7 = channel, and 8 = TcB-binding domain and the linker domain in black. Domains that could not be built are shown as solid circles, and domains that are missing in the sequence are hatched.

  • Fig. 2 Organization of the α-helical shell.

    (A) The α-helical shell of a TcA protomer shown for Pl-TcdA1 can be divided into a small lobe (amino acids 1 to 160, 964 to 1090, 1608 to 1632, and 1762 to 1972) and a large lobe (amino acids 161 to 297 and 434 to 963). The small lobe has a pseudo-twofold symmetry (light and dark blue), resulting in an X-shaped structure. The large lobe contains two pseudo repeats; pseudo repeat 1 is shown in red, orange, and yellow, and pseudo repeat 2 is shown in rose and magenta. (B) The three repeating subdomains of pseudo repeat 1 are depicted in red, orange, and yellow in the upper panel and in rainbow colors (colored from blue to red from N- to C-terminus) in the lower panel. (C) The two repeating subdomains of pseudo repeat 2 are depicted in rose and magenta in the upper panel and in rainbow colors in the lower panel. (D) The pseudo repeat 2 in Yp-TcaATcaB shows the same overall fold, except for the insertion of the coiled-coil domain and an enlarged loop (both in gray).

  • Fig. 3 A 31 trefoil protein knot is present in all TcAs.

    (A) A 31 trefoil protein knot is present in all five TcA prepores. The knot structure in a Pl-TcdA1 prepore protomer and a close-up view is presented. The polypeptide chain is highlighted in rainbow colors. The dashed line indicates missing residues (amino acids 1933 to 1938). (B) The protein knot is also present in the Pl-TcdA1 protomer in the pore state (Protein Data Bank IDs: 5LKH and 5LKI). The dotted line indicates missing residues (amino acids 191 to 1946). (C) Simplified structure of the protein knot of Pl-TcdA1. The arrows indicate the direction of sequence, and the amino acid (aa) numbering corresponds to Pl-TcdA1. (D) Scheme of the shell organization of a TcA protomer showing the location of the knot in the small lobe. RBD A to RBD D and the neuraminidase-like domain are inserted in the main sequence of the big and small lobes.

  • Fig. 4 Comparison of the TcA channels.

    (A) Cross sections of the TcA channels demonstrating the electrostatic Coulomb potential in the channel lumen of Pl-TcdA1, Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB at pH 7. Positively charged (14 kcal/mol) and negatively charged (−14 kcal/mol) residues are colored in blue and red, respectively. (B) Graph depicting the inner channel radius of Pl-TcdA1 (red), Xn-XptA1 (blue), Mm-TcdA4 (orange), and Yp-TcaATcaB (green). At the narrowest position of the Pl-TcdA1 prepore (Y2163, indicated by the dashed line), the diameter of the channel is 3.9 Å. In Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB, the channel diameter reaches only 8.2, 7.8, and 8.4 Å at the narrowest part, respectively. (C) Slice through the channel at the position of the Pl-TcdA1 channel constriction (Y2163).

  • Fig. 5 Formation of chimeric holotoxins.

    (A to D) Negative-stain electron micrographs after complex formation of different TcAs with TcdB2-TccC3 from P. luminescens and size exclusion chromatography. For each complex, a holotoxin particle is highlighted by circles. For Mm-TcdA4 and Yp-TcaATcaB, unbound TcAs are marked with dashed circles. Scale bars, 100 nm. (E) Table with the measured affinities for the chimeric complexes by biolayer interferometry including the dissociation constant (KD) and the on- and off-rate of complex formation (kon and koff, respectively). A global fit according to a 1:1 binding model was applied, including six to seven individual curves. The obtained parameters are the mean value ± the error of the fit. See also fig. S6.

  • Fig. 6 Electrostatic potential of the neuraminidase-like domain and conserved ionic interactions in the shell of TcAs.

    (A to C) Surface electrostatic Coulomb potential of the neuraminidase-like domain at different pH values, viewed from the bottom of TcA. Positively charged (14 kcal/mol) and negatively charged (−14 kcal/mol) residues are colored in blue and red, respectively. Surface electrostatic Coulomb potential at pH 4 (A), pH 7 (B), and pH 11 (C) are shown. The cryo-EM density map of the 99 residues, which could not be built in Yp-TcaATcaB, is depicted in gray. (D to F) Conserved ionic interactions between two protomers in Pl-TcdA1, Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB. The left panel shows two Pl-TcdA1 protomers indicating the different interaction sites, and the right panel presents close-up views of each interaction for Pl-TcdA1 (red), Xn-XptA1 (blue), Mm-TcdA4 (orange), and Yp-TcaATcaB (green). (D) Interaction of a glutamate (protomer A) with an arginine (protomer B). The residue distance is 3.8 Å in the prepore and 9.2 Å in the pore state of Pl-TcdA1. (E) Interaction of an aspartate (protomer A) with an arginine (protomer B). The residue distance is 3.5 Å in the prepore and 5.8 Å in the pore state of Pl-TcdA1. The interacting residues (D and E) belong to the α-helical shell domain. (F) Interaction of an arginine (neuraminidase-like domain of protomer A) with a glutamate (small lobe of protomer B). The residue distance is 3.9 Å in the prepore and 25 Å in the pore state of Pl-TcdA1.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax6497/DC1

    Fig. S1. Structure and conservation of TcAs.

    Fig. S2. Cryo-EM of Pl-TcdA1, Pl-TcdA4, and Xn-XptA1.

    Fig. S3. Cryo-EM of Mm-TcdA4 and Yp-TcaATcaB and purification of Yp-TcaATcaB(WT) and Yp-TcaATcaB–∆622–714.

    Fig. S4. A 31 trefoil protein knot is present in all five TcAs.

    Fig. S5. Biophysical properties of the TcA channels.

    Fig. S6. pH stability of Yp-TcaATcaB and characterization of chimeric holotoxin formation.

    Fig. S7. Intoxication of HEK293T cells with chimeric holotoxins.

    Fig. S8. Topology of neuraminidase-like domain and nonconserved cluster of three histidine residues.

    Fig. S9. pH-induced pore formation of Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB.

    Fig. S10. Mutational studies of Pl-TcdA1.

    Movie S1. Cryo-EM density maps of Pl-TcdA1, Pl-TcdA4, Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB.

    Movie S2. Molecular trefoil knot in Pl-TcdA1.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Structure and conservation of TcAs.
    • Fig. S2. Cryo-EM of Pl-TcdA1, Pl-TcdA4, and Xn-XptA1.
    • Fig. S3. Cryo-EM of Mm-TcdA4 and Yp-TcaATcaB and purification of Yp-TcaATcaB(WT) and Yp-TcaATcaB–∆622–714.
    • Fig. S4. A 31 trefoil protein knot is present in all five TcAs.
    • Fig. S5. Biophysical properties of the TcA channels.
    • Fig. S6. pH stability of Yp-TcaATcaB and characterization of chimeric holotoxin formation.
    • Fig. S7. Intoxication of HEK293T cells with chimeric holotoxins.
    • Fig. S8. Topology of neuraminidase-like domain and nonconserved cluster of three histidine residues.
    • Fig. S9. pH-induced pore formation of Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB.
    • Fig. S10. Mutational studies of Pl-TcdA1.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Cryo-EM density maps of Pl-TcdA1, Pl-TcdA4, Xn-XptA1, Mm-TcdA4, and Yp-TcaATcaB.
    • Movie S2 (.mov format). Molecular trefoil knot in Pl-TcdA1.

    Files in this Data Supplement:

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