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==Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in clostridium difficile toxin A==
==Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in clostridium difficile toxin A==
<StructureSection load='3ho6' size='340' side='right' caption='[[3ho6]], [[Resolution|resolution]] 1.60&Aring;' scene=''>
<StructureSection load='3ho6' size='340' side='right'caption='[[3ho6]], [[Resolution|resolution]] 1.60&Aring;' scene=''>
== Structural highlights ==
== Structural highlights ==
<table><tr><td colspan='2'>[[3ho6]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/"bacillus_difficilis"_hall_and_o'toole_1935 "bacillus difficilis" hall and o'toole 1935]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3HO6 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3HO6 FirstGlance]. <br>
<table><tr><td colspan='2'>[[3ho6]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Clostridioides_difficile Clostridioides difficile]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3HO6 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3HO6 FirstGlance]. <br>
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=IHP:INOSITOL+HEXAKISPHOSPHATE'>IHP</scene></td></tr>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.6&#8491;</td></tr>
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">tcdA, toxA ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1496 "Bacillus difficilis" Hall and O'Toole 1935])</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=IHP:INOSITOL+HEXAKISPHOSPHATE'>IHP</scene></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3ho6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3ho6 OCA], [http://pdbe.org/3ho6 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=3ho6 RCSB], [http://www.ebi.ac.uk/pdbsum/3ho6 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=3ho6 ProSAT]</span></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=3ho6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3ho6 OCA], [https://pdbe.org/3ho6 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3ho6 RCSB], [https://www.ebi.ac.uk/pdbsum/3ho6 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3ho6 ProSAT]</span></td></tr>
</table>
</table>
== Function ==
== Function ==
[[http://www.uniprot.org/uniprot/TOXA_PEPDI TOXA_PEPDI]] Only after the enteral delivery of the enterotoxin A may the characteristic disease called pseudomembranous colitis be induced.  
[https://www.uniprot.org/uniprot/TCDA_CLODI 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]<ref>PMID:16622409</ref> <ref>PMID:1670930</ref> <ref>PMID:17334356</ref> <ref>PMID:19252482</ref> <ref>PMID:19553670</ref> <ref>PMID:20844489</ref> <ref>PMID:27571750</ref> <ref>PMID:27680706</ref> <ref>PMID:31160825</ref>  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).<ref>PMID:22267739</ref> <ref>PMID:22747490</ref> <ref>PMID:24905543</ref> <ref>PMID:30622517</ref> <ref>PMID:7775453</ref>
== Evolutionary Conservation ==
== Evolutionary Conservation ==
[[Image:Consurf_key_small.gif|200px|right]]
[[Image:Consurf_key_small.gif|200px|right]]
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</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=3ho6 ConSurf].
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=3ho6 ConSurf].
<div style="clear:both"></div>
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.
Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A.,Pruitt RN, Chagot B, Cover M, Chazin WJ, Spiller B, Lacy DB J Biol Chem. 2009 Aug 14;284(33):21934-40. Epub 2009 Jun 24. PMID:19553670<ref>PMID:19553670</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 3ho6" style="background-color:#fffaf0;"></div>
== References ==
== References ==
<references/>
<references/>
__TOC__
__TOC__
</StructureSection>
</StructureSection>
[[Category: Bacillus difficilis hall and o'toole 1935]]
[[Category: Clostridioides difficile]]
[[Category: Lacy, D B]]
[[Category: Large Structures]]
[[Category: Pruitt, R N]]
[[Category: Lacy DB]]
[[Category: Enterotoxin]]
[[Category: Pruitt RN]]
[[Category: Inositol phosphate]]
[[Category: Toxin]]

Latest revision as of 13:00, 21 February 2024

Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in clostridium difficile toxin AStructure-function analysis of inositol hexakisphosphate-induced autoprocessing in clostridium difficile toxin A

Structural highlights

3ho6 is a 2 chain structure with sequence from Clostridioides difficile. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 1.6Å
Ligands:
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

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]

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

References

  1. ↑ Greco A, Ho JG, Lin SJ, Palcic MM, Rupnik M, Ng KK. Carbohydrate recognition by Clostridium difficile toxin A. Nat Struct Mol Biol. 2006 May;13(5):460-1. Epub 2006 Apr 16. PMID:16622409 doi:http://dx.doi.org/10.1038/nsmb1084
  2. ↑ Tucker KD, Wilkins TD. Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y. Infect Immun. 1991 Jan;59(1):73-8. doi: 10.1128/iai.59.1.73-78.1991. PMID:1670930 doi:http://dx.doi.org/10.1128/iai.59.1.73-78.1991
  3. ↑ Reineke J, Tenzer S, Rupnik M, Koschinski A, Hasselmayer O, Schrattenholz A, Schild H, von Eichel-Streiber C. Autocatalytic cleavage of Clostridium difficile toxin B. Nature. 2007 Mar 22;446(7134):415-9. doi: 10.1038/nature05622. Epub 2007 Mar 4. PMID:17334356 doi:http://dx.doi.org/10.1038/nature05622
  4. ↑ Lyras D, O'Connor JR, Howarth PM, Sambol SP, Carter GP, Phumoonna T, Poon R, Adams V, Vedantam G, Johnson S, Gerding DN, Rood JI. Toxin B is essential for virulence of Clostridium difficile. Nature. 2009 Apr 30;458(7242):1176-9. doi: 10.1038/nature07822. Epub 2009 Mar 1. PMID:19252482 doi:http://dx.doi.org/10.1038/nature07822
  5. ↑ Pruitt RN, Chagot B, Cover M, Chazin WJ, Spiller B, Lacy DB. Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A. J Biol Chem. 2009 Aug 14;284(33):21934-40. Epub 2009 Jun 24. PMID:19553670 doi:10.1074/jbc.M109.018929
  6. ↑ Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. The role of toxin A and toxin B in Clostridium difficile infection. Nature. 2010 Oct 7;467(7316):711-3. doi: 10.1038/nature09397. Epub 2010 Sep 15. PMID:20844489 doi:http://dx.doi.org/10.1038/nature09397
  7. ↑ Chumbler NM, Rutherford SA, Zhang Z, Farrow MA, Lisher JP, Farquhar E, Giedroc DP, Spiller BW, Melnyk RA, Lacy DB. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016 Jan 11;1:15002. doi: 10.1038/nmicrobiol.2015.2. PMID:27571750 doi:http://dx.doi.org/10.1038/nmicrobiol.2015.2
  8. ↑ Tao L, Zhang J, Meraner P, Tovaglieri A, Wu X, Gerhard R, Zhang X, Stallcup WB, Miao J, He X, Hurdle JG, Breault DT, Brass AL, Dong M. Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature. 2016 Oct 20;538(7625):350-355. doi: 10.1038/nature19799. Epub 2016 Sep, 28. PMID:27680706 doi:http://dx.doi.org/10.1038/nature19799
  9. ↑ Tao L, Tian S, Zhang J, Liu Z, Robinson-McCarthy L, Miyashita SI, Breault DT, Gerhard R, Oottamasathien S, Whelan SPJ, Dong M. Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells. Nat Microbiol. 2019 Oct;4(10):1760-1769. doi: 10.1038/s41564-019-0464-z. Epub, 2019 Jun 3. PMID:31160825 doi:http://dx.doi.org/10.1038/s41564-019-0464-z
  10. ↑ Pruitt RN, Chumbler NM, Rutherford SA, Farrow MA, Friedman DB, Spiller B, Lacy DB. Structural determinants of the Clostridium difficile toxin A glucosyltransferase activity. J Biol Chem. 2012 Jan 20. PMID:22267739 doi:10.1074/jbc.M111.298414
  11. ↑ D'Urzo N, Malito E, Biancucci M, Bottomley MJ, Maione D, Scarselli M, Martinelli M. The structure of Clostridium difficile TcdA-GT domain bound to Mn(2+) and UDP provides insight into glucosyltransferase activity and product release. FEBS J. 2012 Jul 2. doi: 10.1111/j.1742-4658.2012.08688.x. PMID:22747490 doi:10.1111/j.1742-4658.2012.08688.x
  12. ↑ Genth H, Pauillac S, Schelle I, Bouvet P, Bouchier C, Varela-Chavez C, Just I, Popoff MR. Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins. Cell Microbiol. 2014 Nov;16(11):1706-21. doi: 10.1111/cmi.12321. Epub 2014 Aug 4. PMID:24905543 doi:http://dx.doi.org/10.1111/cmi.12321
  13. ↑ Genth H, Junemann J, Lammerhirt CM, Lucke AC, Schelle I, Just I, Gerhard R, Pich A. Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018. Front Microbiol. 2018 Dec 21;9:3078. doi: 10.3389/fmicb.2018.03078. eCollection, 2018. PMID:30622517 doi:http://dx.doi.org/10.3389/fmicb.2018.03078
  14. ↑ Just I, Wilm M, Selzer J, Rex G, von Eichel-Streiber C, Mann M, Aktories K. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J Biol Chem. 1995 Jun 9;270(23):13932-6. doi: 10.1074/jbc.270.23.13932. PMID:7775453 doi:http://dx.doi.org/10.1074/jbc.270.23.13932

3ho6, resolution 1.60Å

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