Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly

Abstract

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (^SymCDTb; 3.14 Å) and an asymmetric form (^AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For ^AsymCDTb, a Ca²⁺ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Keywords: Clostridium difficile; NMR; X-ray crystallography; cryo-EM; structural biology.

Fig. 1.

Functional studies of the RBD2 of CDTb. (A) Cellular toxicity upon the addition of CDTa to Vero cells in the presence of active CDTb (●) or active CDTb lacking the second RBD (i.e., CDTb^∆RBD2; ■). The TC₅₀ of CDTa is 150 ± 40 pM (n = 8 independent experiments, ± SD) when active CDTb is present. Little or no toxicity is observed when CDTa is added Vero cells with active CDTb^∆RBD2 even at the highest concentrations (≥10 nM; n = 3). For simplicity, the x axis presented using the CDTa concentration, but each experiment contains a 7× concentration of active CDTb or CDTb^∆RBD2, as previously described. (B) A representative dominant-negative experiment showing the effect of adding the isolated RBD2 of CDTb (residues 757 to 876) into a Vero cell toxicity assay with 500 pM binary toxin. These data illustrate that the isolated RBD2 inhibits cellular toxicity, as a dominant-negative (IC₅₀ = 20 ± 10 nM; n = 3) calculated using a 4-parameter logistic regression analysis. All data are plotted versus the normalized fluorescence of Alexa Fluor 488 Phalloidin, which selectively labels F-actin filaments; an increase in toxicity causes depolymerization of actin, causing a decrease in fluorescent signal.

Fig. 2.

Biophysical studies of the oligomerization state of activated CDTb. (A) SEC-MALS trace for activated CDTb (red) and the CDTa:CDTb complex (blue). Trace represents absorbance measurements; dots are molecular weight estimates. Representative model structures are shown to designate the corresponding peaks for CDTb. (B) Sedimentation velocity analysis of an 11-µM active CDTb sample indicates that it is predominantly monomeric. (B, Upper) The time-derivative distribution (blue triangles) and the best-fit of the data to a 2 species model (black line). (Lower) Residuals of the fit to a 2-species model. The errors in the sedimentation coefficients [s × (20/w)] and the molecular weights represent the 95% confidence intervals. (C) SAXS curve for activated CDTb. Experimental data (black dots) is shown and fitted with a model that included a mix of ^AsymCDTb and ^SymCDTb at approximately a 1:1 ratio.

Fig. 3.

Structures of activated CDTb. (A) Local resolution in structures of ^AsymCDTb and ^SymCDTb conformations. Increased flexibility is observed in outer regions of the core heptamer, most pronounced for the RBD1 domain. (B) Overall structure of the activated CDTb tetradecamer in ^AsymCDTb and ^SymCDTb conformations. Color scheme is shown in domain diagram and both models are on the same scale, demonstrating slight shortening of the ^AsymCDTb. Domains include a heptamerization domain (HD1; residues 212 to 297), a βBD (residues 298 to 401), a second heptamerization domain (HD2; residues 402 to 486), a linker region (L1; residues 487 to 513), a third heptamerization domain (HD3; residues 514 to 615), an RBD (RBD1; residues 616 to 744), a second linker (L2; residues 745 to 756), and a second RBD (RBD2; residues 757 to 876). The secretion peptide (SP) and the activation domain (AD) are removed via chymotrypsin processing to provide activated CDTb (see also SI Appendix, Figs. S4–S11).

Fig. 4.

Large conformational differences when the 2 heptamer domains of ^SymCDTb without the β-barrel and ^AsymCDTb having the β-barrel are compared. (A) Different packing of the βBD occurs in the 2 different heptamer conformations. For visualization, the single chain of the βBD is highlighted in lighter green. (B) The RBD2 domain donut assembly is located differently in the 2 different heptamer conformations shifts. For clarity, a single polypeptide chain is highlighted in green to show the varied arrangement of the RBD2 domains.

Fig. 5.

Comparison of the “β-barrel” containing heptamer of ^AsymCDTb to the analogous heptamer from the protective antigen (PA) of the anthrax toxin. (A). Heptamers from PA of anthrax toxin are superimposed with electron density from the “β-barrel heptamer” observed in the ^AsymCDTb di-heptamer structure. The RBD in “β-barrel form” of the PA from anthrax toxin were not modeled in the corresponding cryo-EM model and are placed here using alignment with the soluble form of the toxin. (B) Structural comparison of heptameric forms A (Upper) and B (Lower) from ^AsymCDTb (green) and anthrax toxin (red). Cryo-EM densities are shown for all molecules except anthrax toxin form B for which the 2Fo–Fc map is shown and derived from the corresponding crystal structure.

Fig. 6.

Detailed structural features of the active CDTb RBDs. (A) Dual calcium binding site located in the N-terminal region of the protein. The coulomb potential maps (i.e., cryo-EM density maps; blue) in both ^SymCDTb and ^AsymCDTb resolved 2 Ca²⁺ ions bound (Ca1, Ca2; green) with Ca1 oxygen ligands from D222/D224/E231/D273/N260(C = O)/E263(C = O), and Ca²⁺ ligands from D220/D222/D224/E321/D228/I226(C = O). (B) Calcium-binding site located in the β-sandwich domain of RBD1. The RBD1 of CDTb is shown in blue, superimposed with the structure of the β-sandwich from Clostridium thermocellum xylanase Xyn10B used here as an example of Ca²⁺-binding CBM domain (green). (C) Calcium is required for stability of the isolated RBD1. The ¹H,¹⁵N-HSQC spectra of RBD1 are illustrated in the absence (blue) and presence (red) of 6 mM CaCl₂. A large number of the correlations, in the absence of Ca²⁺ (blue) were absent due to exchange-broadening or very strong (marked by “x”) consistent with this construct being “unfolded” in the absence of Ca²⁺. Upon Ca²⁺addition, the backbone and sidechain (i.e., for R668ε) correlations appeared and were highly dispersed, consistent with the RBD1 domain folding in a Ca²⁺-dependent manner. Labeled are resonance assignments for ¹H-¹⁵N correlations (in red) that are fully correlated with their corresponding ¹³C_α and ¹³C_β chemical shift values, along with 96% of C′ shifts, and 93% of the side-chain shift values from triple resonance heteronuclear NMR data. Six other correlations were not assigned (marked with an asterisk, *) due to a complete lack of interresidue correlations in the triple-resonance NMR spectra; perhaps some of these unassigned correlations arise from the 6-residue His-Tag used for purifying this domain. Nine other observable correlations (red; labeled with an “x”) were not assigned, even in the presence of Ca²⁺, and remain disordered based on their narrow line shape and high intensity. Similarly, 29 ¹⁵N-¹H correlations (in blue) for residues of RBD1, in the absence of Ca²⁺, were not be readily assigned due to their intrinsically disordered state. (D) The predicted secondary structure of RBD1 in the presence of Ca²⁺ is predominantly β-strands and consistent with that of RBD1 observed in the cryo-EM structures (Fig. 3 and SI Appendix, Fig. S10), and is comprised of 9 β-strands spanning residues: K615-N621; Y625-N626; G645-P659; K667-D677; S683-A690; E693-P700; T705-T714; N720-G727, and Y732-N742.

Fig. 7.

Large scale structural features of activated CDTb. (A) Cross heptamer hydrophobic interactions between the tip of the βBD in barrel conformation and HD2 for ^AsymCDTb. The non–β-barrel (red) and the β-barrel (green) heptameric assemblies of the di-heptamer are distinguished in different colors for clarity. (B and C) Changes in the size of the pore formed by φ-gate residues (F455) in 2 heptameric forms of the CDTb – ^AsymCDTb (B) and ^SymCDTb (C). In B and C, the color scheme is as defined in Fig. 3 with the phenylalinine residues comprising the pore shown in gray.