Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

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 Å 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 β-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 π-cation interaction within the β-flap that appear to be unique among the large clostridial cytotoxins.Clostridium difficile is a Gram-positive, spore-forming anaerobe that infects the colon and causes a range of disorders, including diarrhea, pseudomembranous colitis, and toxic megacolon (1, 2). Two large toxins, TcdA² and TcdB (308 and 270 kDa, respectively) are recognized as the main virulence factors of C. difficile, although their relative importance is the subject of on-going study (3, 4). These proteins belong to a class of homologous toxins called large clostridial toxins (LCTs) and have been classified more broadly as AB toxins, wherein a B moiety is involved in the delivery of an enzymatic A moiety into the cytosol of a target cell. In LCTs, the A subunit is an N-terminal glucosyltransferase that inactivates small G-proteins, 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 (7–9) and forming the membrane pore needed for translocation of the A subunit (10, 11). Unlike other known AB toxins, the glucosyltransferase A domains of LCTs are released from the B subunits by an autoproteolytic cleavage event (12). Cleavage is triggered by host inositol phosphates and the reducing environment of the cytosol (12).In LCTs, autoproteolysis has been attributed to a cysteine protease activity located within the N-terminal region of the B subunit (13). 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). Autoprocessing in the MARTX toxin from Vibrio cholera (VcRTx) is also stimulated by InsP6 (15). A recent crystal structure of VcRTx CPD bound to InsP6 suggests a novel mechanism of InsP6-induced allosteric activation (16). The CPDs of TcdA and VcRTx share only 19% sequence identity. To gain insight into the mechanistic commonalities between these entirely different toxins and to delineate the LCT-specific modes of InsP6-induced processing, we performed structural and functional analyses on the cysteine protease from TcdA.