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. 2009 Aug 14;284(33):21934-21940.
doi: 10.1074/jbc.M109.018929. Epub 2009 Jun 24.

Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A

Rory N Pruitt  1 , Benjamin Chagot  2 , Michael Cover  1 , Walter J Chazin  2 , Ben Spiller  3 , D Borden Lacy  4
Affiliations

Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A

Rory N Pruitt et al. J Biol Chem. .

Abstract

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.

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Figures

FIGURE 1.
FIGURE 1.
Defining the TcdA CPD. A, TcdA is an AB toxin with four functional domains. The CPD is defined in this work as residues 543–809 based on the InsP6-induced activity of g-CPD, residues 510–809. B, time course of g-CPD (5 μm) cleavage in the presence of 5 μm InsP6 shows 50% cleavage within 10 min. Inset, the band intensities of cleaved and uncleaved proteins were analyzed by densitometry to calculate the percentage of cleavage. The values represent the mean ± S.D. of three independent experiments.
FIGURE 2.
FIGURE 2.
Structure of the TcdA CPD bound to InsP6. TcdA CPD is shown as a ribbon diagram with InsP6 and the side chains of Cys700, His655, Asp589, and Cys597 shown as sticks. The InsP6 binding site and catalytic site are separated by a three-stranded β-flap (blue). The central β-sheet is shown in yellow. The N terminus (red) wraps around the domain with the most N-terminal residues near the catalytic site.
FIGURE 3.
FIGURE 3.
Catalytic site of the TcdA CPD. A, wild-type (WT), C700S, H655A, D589N, and D590N g-CPD at 5 μm were incubated with 100 μm InsP6 for 2 h at 37 °C. In contrast to wild-type and D590N, the C700S, H655A, and D589N mutants were unable to undergo InsP6-inducible cleavage. B, ribbon diagram of the catalytic site of CPD (gray) with residue side chains of the catalytic site (orange) and hydrophobic pocket (green) shown as sticks. Other side chains within the active site are colored gray. C, L542A g-CPD (cleavage site mutant) does not undergo cleavage upon the addition of InsP6. When C700S and L542A g-CPD are mixed, no InsP6-dependent cleavage is observed. D, surface representation of the catalytic site reveals a likely substrate-binding pocket. The Asp589, His655, and Cys700 residues are colored orange, and the hydrophobic residues are colored green. The orientation is identical to that in B.
FIGURE 4.
FIGURE 4.
InsP6 binding pocket. A, the TcdA CPD (left) and VcRTx CPD (right) surfaces colored by electrostatic potential (negatively charged surfaces are red, positively charged regions are blue, and neutral regions are shown in white) demonstrate that InsP6, shown as a stick model, binds in positively charged pockets. The protein molecules were aligned and are shown in identical views, but the orientation of the InsP6 molecules and the position of the axial phosphate groups (denoted with an asterisk) differs. B, InsP6 and the side chains of residues that directly interact with InsP6 are shown as sticks. The orientation is identical to that in A. The displayed electron density is a 2Fo − Fc map contoured at 5σ in which InsP6 was omitted from the phase calculations. C, wild type (WT), K577N, K602N, K754N, and K794N g-CPD at 5 μm were tested for autoproteolysis over a range of InsP6 concentrations. Each mutant required 10–1000-fold more InsP6 in order to undergo autoproteolysis at levels comparable with wild type.
FIGURE 5.
FIGURE 5.
β-flap. A close up of the β-flap (blue) reveals a network of interactions that link InsP6 (red) to the catalytic site (Cys700 and His655).
FIGURE 6.
FIGURE 6.
InsP6-induced structural changes. One-dimensional 1H NMR spectra of the TcdA CPD in the presence (upper trace) and absence (lower trace) of InsP6 show clear dispersion of peaks indicative of folded proteins. The inset shows major changes in the peaks, suggesting that significant structural changes are induced by InsP6 binding.
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