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.
Highlights
Such as Rho, leading to cell rounding and apoptosis of the intoxicated cell [5, 6]
The B subunit corresponds to the remainder of the toxin and is responsible for binding the target cell through a C-terminal receptor-binding domain [7,8,9] and forming the membrane pore needed for translocation of the A subunit [10, 11]
This region was identified based on homology with the cysteine protease domain (CPD) found in the multifunctional autoprocessing repeats in toxins (MARTX) toxins from Gram-negative bacteria [14]
Summary
Plasmid Construction and Point Mutants—The nucleotide sequence coding for amino acids 543– 809 of TcdA (TcdA CPD) was amplified from C. difficile strain 10463 genomic DNA. The DNA was cloned into a modified pET27 vector such that the resulting protein contains an N-terminal His tag followed by a 3C protease cleavage site. The sequence preceding the TcdA CPD sequence is MGSSHHHHHHHHHHGSSLEVLFQGPGS. Following 3C cleavage, only non-native residues GPGS remain. The extended construct, TcdA g-CPD, encodes amino acids 510 – 809. This plasmid was constructed except the last two residues of the leader sequence are VD rather than GS. Point mutants were generated by QuikChange site-directed mutagenesis. Protein Expression and Purification—Transformed Escherichia coli BL21(DE3) cells were grown in Terrific Broth containing 50 mg/liter kanamycin. When the cultures reached A600 ϭ 0.6, the temperature was changed to 16 °C, and expression was induced by the addition of 0.5 mM isopropyl
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