Structure of the full-length Clostridium difficile toxin B

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Abstract

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB–antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

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Fig. 1: Overall structure of the full-length TcdB holotoxin.
Fig. 2: The unique structure of the CROPs of TcdB.
Fig. 3: The CROPs undergoes pH-dependent conformational changes.
Fig. 4: TcdB shows a novel conformation in the pore-forming region at endosomal pH.
Fig. 5: Three modulatory components are clustered at the ‘heart’ of TcdB holotoxin.

Data availability

Atomic coordinates and structure factors of the TcdB–5D–E3–7F, TcdB1072–1433–5D, GTD–E3, and GTDVPI10463–7F complexes have been deposited in the Protein Data Bank under accession codes PDB 6OQ5, PDB 6OQ6, PDB 6OQ7, and PDB 6OQ8, respectively. Source data for Fig. 4e,f are available with the paper online. Other data are available upon request.

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Acknowledgements

This work was partly supported by National Institute of Health grants R01AI139087, R01AI125704, R21AI123920, R21AI139690, and R21CA235533 to R.J., R01GM074830 and R01GM130144 to L.H., R01DK084509, R01AI088748, R01AI132207, and U19AI109776 to H.F, and R01 MH081923 to M.E.B. NE-CAT at the Advanced Photon Source (APS) is supported by a grant from the National Institute of General Medical Sciences (P30GM124165). The Pilatus 6 M detector on 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. DOE, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIH.

Author information

P.C. and R.J. conceived the project. P.C., K.L., Z.L., B.C., and R.J. carried out the protein expression, purification, characterization, crystallization, structure determination and analysis, and all related biochemical studies. K.P. collected the X-ray diffraction data. T.M. carried out the SAXS studies. P.C., C.B.G., and L.H. performed the XL-MS. F.A.M. and M.E.B. carried out the smFRET studies. Y.Z., T.H., and H.F. provided the plasmids of TcdB and VHHs (5D, E3, 7F) and TcdB-expressing Bacillus megaterium. P.C. and R.J. wrote the manuscript with input from other authors.

Correspondence to Rongsheng Jin.

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Competing interests

A provisional patent application has been filed by The Regents of the University of California on the use of the structural information described in this manuscript to prevent and/or treat CDI. R.J. is a cofounder of DesignerT Inc., and H.F. is a co-founder of FZata Inc., which had no role in this study.

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Peer review information: Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Crystal structure of the TcdB–5D–E3–7F complex and the representative electron density maps.

(a) A 2Fo-Fc electron density map for the CROPs (residues G1835–E2367) contoured at 1.0 σ. (b–d) Representative 2Fo-Fc electron density maps for the newly identified SR (residues G1815–S1834) and two other regions in the CROPs (residues G1898–T1927 and G2345–E2367) contoured at 1.0 σ. The bulky residue Tyr was used as one of the markers to confirm the sequence register of the structural model. (e) An anomalous-difference electron density map generated from a crystal of the TcdB–5D–E3–7F complex soaked in tantalum bromide. The map was calculated using a 5.44 Å resolution data collected at λ = 1.2524 Å and phases derived from the final structure model. We found 10 tantalum bromide molecules in one asymmetric unit, which all bind to negatively charged glutamate or aspartate residues and thus confirmed the amino acid register of the complex. One representative tantalum bromide peak was shown in the inset. (f) Overall structure of the TcdB–5D–E3–7F complex. The color scheme for the TcdB domains are the same as that shown in Fig. 1. The three VHHs are colored purple (5D), brown (E3), and green (7F). (g) The structure of TcdB holotoxin is fitted into the negative stain EM map of TcdA that is displayed as a grey surface model (map was generously provided by Dr. Lacy D.B.) (Chumbler, N.M. et al., Nat Microbiol. 1, 15002, 2016). The CROPs of TcdA interacts with the DRBD and adopts a closed conformation.

Supplementary Figure 2 Sequence alignment in the CROPs and the hinge region.

(a) Sequence alignment of all the SRs and LRs in the CROPs of TcdB* (M68 strain), with the sequence conservation displayed on the top of the alignment. (b) Sequence alignment of the hinge region among TcdB*, TcdB (VPI 10463 strain), and TcdB2 (BI/NAP1/027 strain). (c)The hinge region (olive) directly interacts with the 3-HB (blue) and the β-flap (pink). Residues involved in the interactions are colored cyan for the hinge and green for the 3-HB and the β-flap.

Supplementary Figure 3 SEC-SAXS studies on TcdB holotoxin at pH 7.4 and 5.0.

(a, b) Rg (blue) and I(0) (orange) plots for SEC-SAXS at pH 7.4 and 5.0. (c) SAXS profile at pH 7.4 (blue) and pH 5.0 (orange) were superimposed with a curve fitting using the crystal structure of TcdB holotoxin (black). (d) Pairwise distribution function, P(r), of TcdB holotoxin at pH 7.4 (blue) and 5.0 (orange). (e) The maximum dimension of TcdB holotoxin at acidic pH and the core of TcdB composed of GTD, CPD, and DRBD.

Supplementary Figure 4 XL-MS and FRET studies of TcdB holotoxin.

(a) Crosslinking workflow used to unambiguously identify DSSO crosslinks from 3 replicates (Rep1–3) of cross-linked TcdB holotoxin. (b) MSn identification of a DSSO inter-linked peptide from TcdB: K1117–K2249. The cross-linked peptide α–β4+ (m/z 510.9914+) was detected during MS1. Next, it was selected for MS2 where it was fragmented into two characteristic fragment ion pairs, that is αAT (m/z 308.672+/704.312+) and αTA (m/z 324.652+/688.322+). Finally, these peaks were selected for MS3 analysis. αA (m/z 308.672+) identified the sequence DKAATK, in which the lysine residue at position 2 was modified with an alkene moiety. βT (m/z 704.312+) identified the sequence as YYFDEKTGIMR, in which the lysine residue at position 6 was modified with unsaturated thiol moiety. (c) Dynamic light scattering showed a monodispersed peak for TcdB holotoxin at neutral pH. (d) The distribution plot of identified linkages versus their spatial distances measured in the structure of TcdB holotoxin. (e) 5D has no effect on the fluorescence emission of the dye-labeled VHHs and the TcdB–B39–7F complex based on an ensemble FRET study. TcdB holotoxin was incubated with equimolar ratio of the Alexa-555-labeled B39, the Alexa-647-labeled 7F, or both VHHs, with or without 5D at pH 7 or pH 5. Fluorescence emissions upon excitation by 540 nm were measured. Data are the average of duplicated experiments. (f) Fluorescence intensity over time for single molecules of TcdB in complex with dye-labeled VHHs. Alexa 555, donor-labeled B39 (magenta). Alexa-647, acceptor-labeled F7 (cyan). Data was recorded at a frame rate of 10 Hz. Only molecules showing single step photobleaching to baseline for both dyes were included in the analysis. From the magnitude of the anticorrelated bleaching event, we obtained the γ-correction factor for each molecule that allows us to report the absolute FRET efficiencies. FRET was calculated for each frame until acceptor photobleaching. (g) Histogram of average FRET efficiency for each molecule at either pH 5.0 (filled circles, n = 498) or pH 7.0 (open circles, n = 594). This reports a single value for each molecule, which averages out any conformational dynamics. The mean FRET efficiency was unchanged by changing the time binning of the data. (h) Mean smFRET efficiency for TcdB in complex with dye-labeled VHHs at intermediate pH. FRET was calculated from each 100 ms frame of the movie until acceptor photobleaching, compiled into histograms and fit to a Gaussian function to obtain the FRET efficiencies. Error values represent the standard deviation from replicate measurements.

Supplementary Figure 5 VHHs 5D, 7F, and E3 recognize distinct epitopes and exploit different mechanisms to neutralize TcdB.

(a, b) pH-dependent conformational change of TcdB1072–1433 probed by ANS. TcdB1072–1433, the TcdB1072–1433–5D complex, or 5D alone was incubated with ANS in buffers with different pH, and the peak fluorescence intensities of ANS at 474 nm are shown. Error bar represents SD of three replicate experiments. (c) The structure of the TcdB1072–1433–5D complex (light blue, residues 1090–1431 of TcdB were fully resolved in this crystal structure) was superimposed to the full length TcdB–5D complex (TcdB and 5D were colored yellow and pink, respectively). The overall Cα atom r.m.s.d. is ~0.608 Å. (d) Structural superposition to compare the conformations of the pore-forming region among the TcdB1072–1433–5D complex that was crystallized at pH 8.5 (light blue), the TcdB–5D complex that was crystallized at acidic pH (yellow), and TcdA at neutral pH (orange). (e, f) A close-up view into the interface between TcdB and 5D in the structure of the TcdB1072–1433–5D complex, where the CDR2 of 5D (e), as well as its CDR1 and CDR3 (f) bind TcdB with extensive interactions. (g) 7F fixes the conformation of the Helix 1 (residues 525–539) and the Helix 2 (residues 137–158) in the GTD, which are in close proximity to the active site of the CPD and the scissile bond (L544–G545). GTD, CPD, and 7F are colored red, light blue, and green, respectively. The peptide linker between the GTD and the CPD is colored blue. A close-up view of the boxed area is shown in (h). (i) Detailed interactions between GTDVPI10463 and 7F. The interacting residues are colored green and pink for 7F and GTD, respectively. The 7F-binding interfaces are largely identical between GTDVPI10463 and GTD. (j) Interactions between GTD and E3. The residues involved in interactions are colored wheat and pink for E3 and GTD, respectively.

Supplementary Figure 6 Autoprocessing is self-inhibited in TcdB holotoxin.

(a) A close-up view of a zinc atom bound in the active site of the CPD in TcdB holotoxin. (b, c) A simplified cartoon model showing the co-localization of the β-flap, the 3-HB, and the hinge. In TcdB holotoxin, the β-flap is kept in an inactive conformation (pink). Cytosolic InsP6 triggers a ~90° rotation of the β-flap (green) to activate CPD. (d) TcdB1–1805 showed a higher autoprocessing efficiency than the full length TcdB. The autoprocessing of TcdB was examined at 37°C for 1 hour incubation in the presence of various concentrations of InsP6. The results were assessed by SDS-PAGE. 7F potently inhibited GTD cleavage. The molecular weight of TcdB holotoxin, TcdB1–1805, GTD, and 7F is 270 kDa, 205 kDa, 64 kDa, and 15.2 kDa, respectively. The gel is representative of three independent experiments.

Supplementary Figure 7 Mapping the binding sites for CSPG4, FZDs, and bezlotoxumab in TcdB holotoxin.

(a) The reported CSPG4-binding site in TcdB, involving residues 1500–1900 (Gupta, P. et al., J Biol Chem. 292, 17290-17301, 2017 and Yuan, P. et al., Cell Res. 25, 157-68, 2015), was colored blue. (b) Binding of TcdB to the immobilized biotin-labeled CRD2 was examined using a pull-down assay at neutral and acidic pH. Samples were analyzed by SDS-PAGE and Coomassie Blue staining. (c) The structure of the cysteine-rich domain of FZD2 (CRD2, green cartoon model) in complex with TcdB1285–1804 (PDB code: 6C0B) (Chen, P. et al., Science, 360, 664-669, 2018) was superimposed to TcdB holotoxin. The pore-forming region of TcdB is shown in a purple ribbon model while the rest of the toxin is shown in a surface model. (d) The structure of the CROPs I–II of TcdB in complex with bezlotoxumab Fabs (PDB code: 4NP4) (Orth, P. et al., J Biol Chem, 289, 18008-21, 2014) was superimposed to TcdB holotoxin. Bezlotoxumab Fabs were shown as cartoon models, which bind to two sites on the CROPs (blue and pink). Bezlotoxumab clashes with the GTD and the DRBD when TcdB adopts the acidic conformation. A close-up view of the boxed area is shown in (e).

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Tables 1–3, and Supplementary Note 1

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Source Data, Fig. 4e,f

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