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[[Image: | ==RICIN A-CHAIN (RECOMBINANT) E177D MUTANT== | ||
<StructureSection load='2vc4' size='340' side='right' caption='[[2vc4]], [[Resolution|resolution]] 1.39Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[2vc4]] is a 1 chain structure with sequence from [http://en.wikipedia.org/wiki/Ricinus_communis Ricinus communis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2VC4 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2VC4 FirstGlance]. <br> | |||
</td></tr><tr><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene><br> | |||
<tr><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[1apg|1apg]], [[1br5|1br5]], [[1br6|1br6]], [[1j1m|1j1m]], [[1obs|1obs]], [[1obt|1obt]], [[1uq4|1uq4]], [[1zam|1zam]], [[1zb2|1zb2]], [[2aai|2aai]], [[1fmp|1fmp]], [[1ifs|1ifs]], [[1ift|1ift]], [[1ifu|1ifu]], [[1il3|1il3]], [[1il4|1il4]], [[1il5|1il5]], [[1il9|1il9]], [[1rtc|1rtc]], [[1uq5|1uq5]], [[1zb0|1zb0]], [[2vc3|2vc3]]</td></tr> | |||
<tr><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/rRNA_N-glycosylase rRNA N-glycosylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.2.22 3.2.2.22] </span></td></tr> | |||
<tr><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2vc4 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2vc4 OCA], [http://www.rcsb.org/pdb/explore.do?structureId=2vc4 RCSB], [http://www.ebi.ac.uk/pdbsum/2vc4 PDBsum]</span></td></tr> | |||
<table> | |||
== Evolutionary Conservation == | |||
[[Image:Consurf_key_small.gif|200px|right]] | |||
Check<jmol> | |||
<jmolCheckbox> | |||
<scriptWhenChecked>select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/vc/2vc4_consurf.spt"</scriptWhenChecked> | |||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | |||
<text>to colour the structure by Evolutionary Conservation</text> | |||
</jmolCheckbox> | |||
</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/chain_selection.php?pdb_ID=2ata ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed. | |||
The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae.,Allen SC, Moore KA, Marsden CJ, Fulop V, Moffat KG, Lord JM, Ladds G, Roberts LM FEBS J. 2007 Nov;274(21):5586-99. Epub 2007 Oct 4. PMID:17916187<ref>PMID:17916187</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[Ricin|Ricin]] | *[[Ricin|Ricin]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Ricinus communis]] | [[Category: Ricinus communis]] | ||
[[Category: RRNA N-glycosylase]] | [[Category: RRNA N-glycosylase]] | ||
Revision as of 09:52, 29 September 2014
RICIN A-CHAIN (RECOMBINANT) E177D MUTANTRICIN A-CHAIN (RECOMBINANT) E177D MUTANT
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedRicin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed. The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae.,Allen SC, Moore KA, Marsden CJ, Fulop V, Moffat KG, Lord JM, Ladds G, Roberts LM FEBS J. 2007 Nov;274(21):5586-99. Epub 2007 Oct 4. PMID:17916187[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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