title: Crystal structure ofClostridium difficiletoxin A.
abstract: Clostridium difficileinfection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.
authors: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors: Nicole M
authors: NM
authors: Chumbler
authors: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors: Stacey A
authors: SA
authors: Rutherford
authors: Department of Biochemistry, University of Toronto and the Molecular Structure & Function Research Institute at The Hospital for Sick Children, Toronto, Ontario M5S 1A8, Canada.
authors: Zhifen
authors: Z
authors: Zhang
authors: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors: Melissa A
authors: MA
authors: Farrow
authors: Interdisciplinary Graduate Program in Biochemistry, Indiana University, Bloomington, Indiana 47405, USA; Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
authors: John P
authors: JP
authors: Lisher
authors: Case Western Reserve University Center for Synchrotron Biosciences, National Synchrotron Light Source, Building 725, Brookhaven National Laboratory, New York 11973, USA.
authors: Erik
authors: E
authors: Farquhar
authors: Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
authors: David P
authors: DP
authors: Giedroc
authors: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37212, USA.
authors: Benjamin W
authors: BW
authors: Spiller
authors: Department of Biochemistry, University of Toronto and the Molecular Structure & Function Research Institute at The Hospital for Sick Children, Toronto, Ontario M5S 1A8, Canada.
authors: Roman A
authors: RA
authors: Melnyk
authors: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37205, USA; The Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, USA.
authors: D Borden
authors: DB
authors: Lacy