Structural Basis for Substrate Recognition in the Enzymatic Component of ADP-ribosyltransferase Toxin CDTa from Clostridium difficile

Abstract

ADP-ribosylation is one of the favored modes of cell intoxication employed by several bacteria. Clostridium difficile is recognized to be an important nosocomial pathogen associated with considerable morbidity and attributable mortality. Along with its two well known toxins, Toxin A and Toxin B, it produces an ADP-ribosylating toxin that targets monomeric actin of the target cell. Like other Clostridial actin ADP-ribosylating toxins, this binary toxin, known as C. difficile toxin (CDT), is composed of two subunits, CDTa and CDTb. In this study, we present high resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5, and 9.0) and in complex with ADP-ribose donors, NAD and NADPH (at pH 9.0). The crystal structures of the native protein show "pronounced conformational flexibility" confined to the active site region of the protein and "enhanced" disorder at low pH, whereas the complex structures highlight significant differences in "ligand specificity" compared with the enzymatic subunit of a close homologue, Clostridium perfringens iota toxin. Specifically in CDTa, two of the suggested catalytically important residues (Glu-385 and Glu-387) seem to play no role or a less important role in ligand binding. These structural data provide the first detailed information on protein-donor substrate complex stabilization in CDTa, which may have implications in understanding CDT recognition.Clostridium difficile infection is a major problem as a healthcare-associated infection. The bacterium causes nosocomial, antibiotic-associated diarrhea and pseudomembranous colitis in patients treated with broad spectrum antibiotics (1-3). Elderly patients are most at risk from these potentially life-threatening diseases, and incidents of hospital infection have increased dramatically over the last 10 years.Strains of C. difficile produce a variety of virulence factors, notable among which are several protein toxins: Toxin A, Toxin B (4 -6), and, in some strains, the binary toxin CDT, 3 which is similar to Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin (7-9). Toxins A and B are large protein cytotoxins that play a key role in the pathology of infection and most probably are involved in the gut colonization process. Outbreaks of C. difficile infection have been reported with Toxin A-negative/Toxin B-positive strains, and a recent report (10) suggests that Toxin B plays a major role in the disease pathology. Little is presently known about the contribution of the binary toxin to C. difficile infection.CDT binary toxin belongs to the family of actin-specific ADP-ribosylating toxin (ADPRT) (for a recent review see Ref.11), composed of two independently produced components: a transport component of 99 kDa (CDTb) that facilitates translocation of the enzymatic component of 49 kDa (CDTa) into the target cell that is capable of transferring ADP-ribose group of NAD/NADPH to monomeric actin molecules in target cells (9,12,13). This irreversible modification of G-actin at 14) blocks its polymerization and thus...

<i>Clostridium difficile</i> Toxins: Mediators of Inflammation

“…The structure of CDTa is similar to the enzymatic domain of C. perfringens Iota toxin, although they exhibit slight differences in their mode of ligand recognition [67] . Like other ADP-ribosylating toxins, the CDTa-CDTb complex induces cell rounding and cell death in Vero cells [68,69] , and the uptake of CDT into cells also requires endosomal acidification [70] .…”

ADP-ribosylating toxins modifying the actin cytoskeleton

“…These enzymes consist of two distinct, but related, structural domains that were probably derived by duplication of an ancestral gene [29,30,76,88]. Each domain consists of a perpendicular packing of five mixed, β-sheet strands against a three-stranded, antiparallel β-sheet flanked by four consecutive helices.…”

Inhibitors of pore-forming toxins

“…While the anthrax toxin is a tripartite toxin made by the two enzymatically active A components and one binding/translocation B component, clostridial binary toxins are made of two components only [24,42]. In contrast to that of the anthrax toxin, the enzymatic A components of clostridial binary toxins act through mono-ADP-ribosylation of G-actins, causing a complete destruction of the actin cytoskeleton and caspase-dependent cell death [19,[51][52][53][54][55][56][57][58][59]. X-ray crystallography of both the PA component of anthrax toxin and the B component of the C2 toxin (C2II) have shown four distinct domains involved in cellular receptor binding, oligomerization, pore formation, and A component binding [30,60].…”