Crystal structure of Clostridium difficile toxin A

Abstract

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

Figure 1. Structure of TcdA

a, The TcdA primary structure can be divided into four functional domains: the glucosyltransferase domain (GTD, red), the autoprotease domain (APD, purple; including the three-helix bundle, dark purple), the delivery domain (yellow) and the CROPS domain (white). b, Cartoon representation of the TcdA₁₈₃₂ structure (coloured according to a), with zinc shown in green. c, The structure in b, rotated 90°, with the GTD shown as a surface view with the UDP–glucose binding site in green. d, The TcdA₁₈₃₂ structure was fit in the 20 Å EM map of TcdA holotoxin using Chimera. The InsP6 binding site is shown in green, and positions for the first and last residues visible in the structure are indicated.

Figure 2. Zinc is required for autoprocessing activity

a, The APD, along with a small portion of the GTD and the three-helix bundle from the TcdA₁₈₃₂ structure (oriented as in Fig. 1a), is depicted with residue 542 in red, residues 543–745 in white, the 746–765 β-flap in light blue, and some of the three-helix bundle (766–801) in dark blue. Zinc (green) is bound in the APD active site by H655, C700 and H759. Four lysines form the initial binding site for InsP6: K602, K649, K754 and K777. b, On comparison with a, the InsP6-bound structure of the TcdA APD (Protein Data Bank: 3HO6) suggests significant structural changes occur with InsP6 binding: the accumulation of eight lysines and one arginine in the InsP6-binding site, a rearrangement of the β-flap and elements of the three-helix bundle, and displacement of H759 from the active site. c, Mutation of TcdA His759 or TcdB His757 leads to proteins that undergo autoprocessing at lower concentrations of InsP6. Cleaved GTD was quantified relative to the holotoxin in the 0 mM InsP6 control, and means ± s.d. (n = 3) are shown. d, Chelation of zinc through treatment with 10 mM TPEN renders TcdA and TcdB incapable of InsP6-induced autoprocessing. e, Chelation of zinc in mutants that show enhanced autoprocessing renders TcdA and TcdB incapable of InsP6-induced autoprocessing. Experiments in d,e were conducted in the presence of 5% ethanol, a solvent for TPEN. f, Autoprocessing can be restored in TPEN-treated autoprocessing-defective preparations of TcdA and TcdB with the addition of ZnCl₂. Gels in c–f are representative of three independent experiments.

Figure 3. The delivery domain provides an extended scaffold for an α-helical hydrophobic stretch involved in pore formation

a, Most of the TcdA₁₈₃₂ crystal structure (residues 1–1025 and 1136–1802) is depicted as a transparent surface with the GTD in white and the APD in blue. The delivery domain is visible as a cartoon to highlight the three-helix bundle (blue), the globular sub-domain (green), the α-helical hydrophobic stretch (residues 1026–1135, pink) and the β-scaffold (yellow). Residues implicated in TcdB pore formation are shown as orange or red sticks. b, Representative sequences from the six large clostridial glycosylating toxins were aligned and scored with a Risler matrix according to the extent of sequence variation. Scores are displayed on the TcdA₁₈₃₂ structure surface with a colour ramp (red, orange, yellow, green, light blue, dark blue) in which strictly conserved residues are coloured red and the most variable residues are coloured dark blue. The most conserved surface region (boxed) is at the end of the α-helical hydrophobic stretch: the 1098–1118 loop and β-hairpin. Within this region, the V1109, N1110 and N1111 residues are notable in their accessibility to solvent. c, The TcdA_SAS protein binds cells at levels equivalent to wild type but is impaired in its capacity to glucosylate Rac1. Toxins (10 nM) were applied to HeLa cells and incubated for 3 h at 37 °C. Proteins were separated by SDS-PAGE and probed with antibodies that recognize TcdA CROPS, non-glucosylated Rac1 or total Rac1. Quantitation of four gels indicates that while 100% of the detectable Rac1 was glucosylated by TcdA, only 23.4 ± 10.8% was glucosylated by TcdA_SAS (relative to mock treated). d, The TcdA_SAS protein is not impaired in its capacity to glucosylate Rac1 in vitro using purified proteins. Toxins (100 nM) were incubated with purified GST-Rac1 for 3 h at 37 °C and analysed as in c. The gel is representative of three independent experiments. e, The TcdA_SAS mutant is defective in its cellular toxicity. Toxins (10 fM–20 nM) were incubated with CHO cells for 48 h at 37 °C and viability was normalized to untreated cells. A representative dose–response curve is shown and values of the effective concentration conferring half maximal protection (EC₅₀) ± s.d. were calculated from two biological replicates using Prism: TcdA (blue circles, EC₅₀ = 0.11 ± 0.01 nM); TcdA_DXD (a glucosyltransferase-defective mutant; orange squares, EC₅₀ > 20 nM); TcdA_SAS, (purple triangles, EC₅₀ = 1.74 ± 0.39 nM). f, Pore formation on biological membranes. TcdA, TcdA_DXD and TcdA_SAS were applied to Vero cells preloaded with ⁸⁶Rb⁺ and then subjected to external medium at pH 4.8. Data represent the means and s.d. associated with four experiments. Colours are as in e.