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. 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 (1Lyerly D.M. Krivan H.C. Wilkins T.D. Clin. Microbiol. Rev. 1988; 1: 1-18Crossref PubMed Scopus (439) Google Scholar, 2McFarland L.V. Stamm W.E. Am. J. Infect. Control. 1986; 14: 99-109Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 3Knoop F.C. Owens M. Crocker I.C. Clin. Microbiol. Rev. 1993; 6: 251-265Crossref PubMed Scopus (148) Google Scholar). 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 (4Lyerly D.M. Saum K.E. MacDonald D.K. Wilkins T.D. Infect. Immun. 1985; 47: 349-352Crossref PubMed Google Scholar, 5Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (882) Google Scholar, 6Voth D.E.