2f9p: Difference between revisions

Revision as of 18:19, 21 February 2008

Crystal Structure of the Recombinant Human Alpha I Tryptase Mutant D216G in Complex with Leupeptin

OverviewOverview

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 StructureAbout this Structure

2F9P is a Single protein structure of sequence from Homo sapiens with , and as ligands. Active as Tryptase, with EC number 3.4.21.59 Full crystallographic information is available from OCA.

ReferenceReference

X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->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

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-[[Image:2f9p.gif|left|200px]]<br /><applet load="2f9p" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="2f9p, resolution 2.30&Aring;" />
 '''Crystal Structure of the Recombinant Human Alpha I Tryptase Mutant D216G in Complex with Leupeptin'''<br />
 ==Overview==
-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.
+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==
-F9P is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with NAG, ACE and BU3 as [http://en.wikipedia.org/wiki/ligands ligands]. 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=2F9P OCA].
+F9P is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=NAG:'>NAG</scene>, <scene name='pdbligand=ACE:'>ACE</scene> and <scene name='pdbligand=BU3:'>BU3</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. 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://oca.weizmann.ac.il/oca-bin/ocashort?id=2F9P OCA].
 ==Reference==
@@ Line 17: / Line 17: @@
 [[Category: Huber, R.]]
 [[Category: Marquardt, U.]]
-[[Category: Rohr, K.B.]]
+[[Category: Rohr, K B.]]
-[[Category: Schechter, N.M.]]
+[[Category: Schechter, N M.]]
 [[Category: Selwood, T.]]
-[[Category: Than, M.E.]]
+[[Category: Than, M E.]]
 [[Category: ACE]]
 [[Category: BU3]]
@@ Line 29: / Line 29: @@
 [[Category: trypsin-like]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 10:26:20 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:19:10 2008''