title: The structure of Clostridium difficile toxin A glucosyltransferase domain bound to Mn2+ and UDP provides insights into glucosyltransferase activity and product release.
abstract: Clostridiumdifficile toxin A (TcdA) is a member of the large clostridial toxin family, and is responsible, together with C. difficile toxin B (TcdB), for many clinical symptoms d ring human infections. Like other large clostridial toxins, TcdA catalyzes the glucosylation of GTPases, and is able to inactivate small GTPases within the host cell. Here, we report the crystal structures of the TcdA glucosyltransferase domain (TcdA-GT) in the apo form and in the presence of Mn(2+) and hydrolyzed UDP-glucose. These structures, together with the recently reported crystal structure of TcdA-GT bound to UDP-glucose, provide a detailed understanding of the conformational changes of TcdA that occur during the catalytic cycle. Indeed, we present a new intermediate conformation of a so-called 'lid' loop (residues 510-522 in TcdA), concomitant with the absence of glucose in the catalytic domain. The recombinant TcdA was expressed in Brevibacillus in the inactive apo form. High thermal stability of wild-type TcdA was observed only after the addition of both Mn(2+) and UDP-glucose. The glucosylhydrolase activity, which is readily restored after reconstitution with both these cofactors, was similar to that reported for TcdB. Interestingly, we found that ammonium, like K(+) , is able to activate the UDP-glucose hydrolase activities of TcdA. Consequently, the presence of ammonium in the crystallization buffer enabled us to obtain the first crystal structure of TcdA-GT bound to the hydrolysis product UDP.
authors: Novartis Vaccines and Diagnostics, Siena, Italy.
authors: Nunzia
authors: N
authors: D'Urzo
authors: Enrico
authors: E
authors: Malito
authors: Marco
authors: M
authors: Biancucci
authors: Matthew J
authors: MJ
authors: Bottomley
authors: Domenico
authors: D
authors: Maione
authors: Maria
authors: M
authors: Scarselli
authors: Manuele
authors: M
authors: Martinelli
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D016360:Clostridioides difficile / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D016695:Glycosyltransferases / Q000378:metabolism*; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014530:Uridine Diphosphate / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D000644:Quaternary Ammonium Compounds; C041348:tcdA protein, Clostridium difficile; D008345:Manganese; D014530:Uridine Diphosphate; D016695:Glycosyltransferases