4dmw
From Proteopedia
Crystal structure of the GT domain of Clostridium difficile toxin A (TcdA) in complex with UDP and Manganese
Structural highlights
Function[TCDA_CLODI] Precursor of a cytotoxin that targets and disrupts the colonic epithelium, inducing the host inflammatory and innate immune responses and resulting in diarrhea and pseudomembranous colitis (PubMed:20844489). TcdA and TcdB constitute the main toxins that mediate the pathology of C.difficile infection, an opportunistic pathogen that colonizes the colon when the normal gut microbiome is disrupted (PubMed:19252482, PubMed:20844489). Compared to TcdB, TcdA is less virulent and less important for inducing the host inflammatory and innate immune responses (PubMed:19252482). This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdA) into the host cytosol (By similarity). Targets colonic epithelia by binding to some receptor, and enters host cells via clathrin-mediated endocytosis (By similarity). Binding to LDLR, as well as carbohydrates and sulfated glycosaminoglycans on host cell surface contribute to entry into cells (PubMed:1670930, PubMed:31160825, PubMed:16622409). In contrast to TcdB, Frizzled receptors FZD1, FZD2 and FZD7 do not act as host receptors in the colonic epithelium for TcdA (PubMed:27680706). Once entered into host cells, acidification in the endosome promotes the membrane insertion of the translocation region and formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (By similarity). This activates the peptidase C80 domain and autocatalytic processing, releasing the N-terminal part (Glucosyltransferase TcdA), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356, PubMed:19553670, PubMed:27571750).[UniProtKB:P18177][1] [2] [3] [4] [5] [6] [7] [8] [9] Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:7775453, PubMed:24905543, PubMed:30622517, PubMed:22747490, PubMed:22267739). Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, Rap2A and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-alpha-D-glucose as the sugar donor (PubMed:7775453, PubMed:24905543, PubMed:30622517, PubMed:22747490, PubMed:22267739). Monoglucosylation of host small GTPases completely prevents the recognition of the downstream effector, blocking the GTPases in their inactive form, leading to actin cytoskeleton disruption and cell death, resulting in the loss of colonic epithelial barrier function (PubMed:7775453). Also able to catalyze monoglucosylation of some members of the Ras family (H-Ras/HRAS, K-Ras/KRAS and N-Ras/NRAS), but with much less efficiency than with Rho proteins, suggesting that it does not act on Ras proteins in vivo (PubMed:30622517).[10] [11] [12] [13] [14] Publication Abstract from PubMedToxin 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[15] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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