4dmw

Publication Abstract from PubMed

Toxin A (TcdA) from Clostridium difficile is part of the Large Clostridium Toxin (LCT) family and is responsible, together with Toxin B, for many clinical symptoms during human infections. Like other LCTs, TcdA catalyses the glucosylation of GTPases and is able to inactivate small GTPases within the host cell. Here, we report the crystal structures of the TcdA-GT domain in the apo-form and in the presence of manganese and hydrolyzed UDP-glucose. These structures, together with the recently reported crystal structure of TcdA-GT domain 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. A high thermal stabilization of WT TcdA was observed only after the addition of both manganese 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, similarly to potassium, is able to activate the UDP-glucose hydrolase activities of TcdA. Consequently, the presence of ammonium in the crystallization buffer enabled us to obtain first crystal structure of the TcdA-GT domain bound to the hydrolysis product UDP. Database: Coordinates of apo-TcdA-GT and Mn(2+) -UDP-TcdA-GT have been deposited in the Protein Data Bank with accession codes 4DMV and 4DMW, respectively.

The structure of Clostridium difficile TcdA-GT domain bound to Mn(2+) and UDP provides insight into glucosyltransferase activity and product release.,D'Urzo N, Malito E, Biancucci M, Bottomley MJ, Maione D, Scarselli M, Martinelli M FEBS J. 2012 Jul 2. doi: 10.1111/j.1742-4658.2012.08688.x. PMID:22747490^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.