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New page: left|200px<br /> <applet load="2f9o" size="450" color="white" frame="true" align="right" spinBox="true" caption="2f9o, resolution 2.10Å" /> '''Crystal Structure o...
 
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[[Image:2f9o.gif|left|200px]]<br />
<applet load="2f9o" size="450" color="white" frame="true" align="right" spinBox="true"
caption="2f9o, resolution 2.10&Aring;" />
'''Crystal Structure of the Recombinant Human Alpha I Tryptase Mutant D216G'''<br />


==Overview==
==Crystal Structure of the Recombinant Human Alpha I Tryptase Mutant D216G==
Tryptases alpha and beta are trypsin-like serine proteinases expressed in, large amounts by mast cells. Beta-tryptase is a tetramer that has, enzymatic activity, but requires heparin binding to maintain functional, and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As, shown previously, these differences can be mainly attributed to the, different conformations of the 214-220 segment. Interestingly, the, replacement of Asp216 by Gly, which is present in beta-tryptase, results, in enzymatically active but less stable alpha-tryptase mutants. We have, solved the crystal structures of both the single (D216G) and the double, (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex, with the peptide inhibitor leupeptin, as well as the structure of the, non-inhibited single mutant. The inhibited mutants exhibited an open, functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like), conformations were present simultaneously. This shows that both forms are, in a two-state equilibrium, which is influenced by the residues in the, vicinity of the active site and by inhibitor/substrate binding. Novel, insights regarding the observed stability differences as well as a, potential proteolytic activity of wild-type alpha-tryptase, which may, possess a cryptic active site, are discussed.
<StructureSection load='2f9o' size='340' side='right'caption='[[2f9o]], [[Resolution|resolution]] 2.10&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2F9O OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2F9O FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.1&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=FUC:ALPHA-L-FUCOSE'>FUC</scene>, <scene name='pdbligand=NAG:N-ACETYL-D-GLUCOSAMINE'>NAG</scene></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=2f9o FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2f9o OCA], [https://pdbe.org/2f9o PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2f9o RCSB], [https://www.ebi.ac.uk/pdbsum/2f9o PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2f9o ProSAT]</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/f9/2f9o_consurf.spt"</scriptWhenChecked>
    <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.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/main_output.php?pdb_ID=2f9o ConSurf].
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.


==About this Structure==
X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha--&gt;beta-tryptase transition.,Rohr KB, Selwood T, Marquardt U, Huber R, Schechter NM, Bode W, Than ME J Mol Biol. 2006 Mar 17;357(1):195-209. Epub 2005 Dec 28. PMID:16414069<ref>PMID:16414069</ref>
2F9O is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Active as [http://en.wikipedia.org/wiki/Tryptase Tryptase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.59 3.4.21.59] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2F9O OCA].


==Reference==
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha--&gt;beta-tryptase transition., Rohr KB, Selwood T, Marquardt U, Huber R, Schechter NM, Bode W, Than ME, J Mol Biol. 2006 Mar 17;357(1):195-209. Epub 2005 Dec 28. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=16414069 16414069]
</div>
[[Category: Homo sapiens]]
<div class="pdbe-citations 2f9o" style="background-color:#fffaf0;"></div>
[[Category: Single protein]]
[[Category: Tryptase]]
[[Category: Bode, W.]]
[[Category: Huber, R.]]
[[Category: Marquardt, U.]]
[[Category: Rohr, K.B.]]
[[Category: Schechter, N.M.]]
[[Category: Selwood, T.]]
[[Category: Than, M.E.]]
[[Category: difucosylation]]
[[Category: serine proteinase]]
[[Category: trypsin-like]]


''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 22:02:06 2007''
==See Also==
*[[Tryptase|Tryptase]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Bode W]]
[[Category: Huber R]]
[[Category: Marquardt U]]
[[Category: Rohr KB]]
[[Category: Schechter NM]]
[[Category: Selwood T]]
[[Category: Than ME]]
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