1ezu: Difference between revisions

Revision as of 13:33, 21 February 2008

ECOTIN Y69F, D70P BOUND TO D102N TRYPSIN

OverviewOverview

Ecotin is a dimeric serine protease inhibitor from Escherichia coli which binds proteases to form a hetero-tetramer with three distinct interfaces: an ecotin-ecotin dimer interface, a larger primary ecotin-protease interface, and a smaller secondary ecotin-protease interface. The contributions of these interfaces to binding and inhibition are unequal. To investigate the contribution and adaptability of each interface, we have solved the structure of two mutant ecotin-trypsin complexes and compared them to the structure of the previously determined wild-type ecotin-trypsin complex. Wild-type ecotin has an affinity of 1 nM for trypsin, while the optimized mutant, ecotin Y69F, D70P, which was found using phage display technologies, inhibits rat trypsin with a K(i) value of 0.08 nM. Ecotin 67-70A, M84R which has four alanine substitutions in the ecotin-trypsin secondary binding site, along with the M84R mutation at the primary site, has a K(i) value against rat trypsin of 0.2 nM. The structure of the ecotin Y69F, D70P-trypsin complex shows minor structural changes in the ecotin-trypsin tetramer. The structure of the ecotin 67-70A, M84R mutant bound to trypsin shows large deviations in the tertiary and quaternary structure of the complex. The trypsin structure shows no significant changes, but the conformation of several loop regions of ecotin are altered, resulting in the secondary site releasing its hold on trypsin. The structure of several regions previously considered to be rigid is also significantly modified. The inherent flexibility of ecotin allows it to accommodate these mutations and still maintain tight binding through the compromises of the protein-protein interfaces in the ecotin-trypsin tetramer. A comparison with two recently described ecotin-like genes from other bacteria suggests that these structural and functional features are conserved in otherwise distant bacterial lineages.

About this StructureAbout this Structure

1EZU is a Protein complex structure of sequences from Escherichia coli and Rattus norvegicus with as ligand. Active as Trypsin, with EC number 3.4.21.4 Full crystallographic information is available from OCA.

ReferenceReference

Compromise and accommodation in ecotin, a dimeric macromolecular inhibitor of serine proteases., Gillmor SA, Takeuchi T, Yang SQ, Craik CS, Fletterick RJ, J Mol Biol. 2000 Jun 16;299(4):993-1003. PMID:10843853

Page seeded by OCA on Thu Feb 21 12:33:18 2008

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-[[Image:1ezu.jpg|left|200px]]<br /><applet load="1ezu" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1ezu.jpg|left|200px]]<br /><applet load="1ezu" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1ezu, resolution 2.40&Aring;" />
 '''ECOTIN Y69F, D70P BOUND TO D102N TRYPSIN'''<br />
 ==Overview==
-Ecotin is a dimeric serine protease inhibitor from Escherichia coli which, binds proteases to form a hetero-tetramer with three distinct interfaces:, an ecotin-ecotin dimer interface, a larger primary ecotin-protease, interface, and a smaller secondary ecotin-protease interface. The, contributions of these interfaces to binding and inhibition are unequal., To investigate the contribution and adaptability of each interface, we, have solved the structure of two mutant ecotin-trypsin complexes and, compared them to the structure of the previously determined wild-type, ecotin-trypsin complex. Wild-type ecotin has an affinity of 1 nM for, trypsin, while the optimized mutant, ecotin Y69F, D70P, which was found, using phage display technologies, inhibits rat trypsin with a K(i) value, of 0.08 nM. Ecotin 67-70A, M84R which has four alanine substitutions in, the ecotin-trypsin secondary binding site, along with the M84R mutation at, the primary site, has a K(i) value against rat trypsin of 0.2 nM. The, structure of the ecotin Y69F, D70P-trypsin complex shows minor structural, changes in the ecotin-trypsin tetramer. The structure of the ecotin, 67-70A, M84R mutant bound to trypsin shows large deviations in the, tertiary and quaternary structure of the complex. The trypsin structure, shows no significant changes, but the conformation of several loop regions, of ecotin are altered, resulting in the secondary site releasing its hold, on trypsin. The structure of several regions previously considered to be, rigid is also significantly modified. The inherent flexibility of ecotin, allows it to accommodate these mutations and still maintain tight binding, through the compromises of the protein-protein interfaces in the, ecotin-trypsin tetramer. A comparison with two recently described, ecotin-like genes from other bacteria suggests that these structural and, functional features are conserved in otherwise distant bacterial lineages.
+Ecotin is a dimeric serine protease inhibitor from Escherichia coli which binds proteases to form a hetero-tetramer with three distinct interfaces: an ecotin-ecotin dimer interface, a larger primary ecotin-protease interface, and a smaller secondary ecotin-protease interface. The contributions of these interfaces to binding and inhibition are unequal. To investigate the contribution and adaptability of each interface, we have solved the structure of two mutant ecotin-trypsin complexes and compared them to the structure of the previously determined wild-type ecotin-trypsin complex. Wild-type ecotin has an affinity of 1 nM for trypsin, while the optimized mutant, ecotin Y69F, D70P, which was found using phage display technologies, inhibits rat trypsin with a K(i) value of 0.08 nM. Ecotin 67-70A, M84R which has four alanine substitutions in the ecotin-trypsin secondary binding site, along with the M84R mutation at the primary site, has a K(i) value against rat trypsin of 0.2 nM. The structure of the ecotin Y69F, D70P-trypsin complex shows minor structural changes in the ecotin-trypsin tetramer. The structure of the ecotin 67-70A, M84R mutant bound to trypsin shows large deviations in the tertiary and quaternary structure of the complex. The trypsin structure shows no significant changes, but the conformation of several loop regions of ecotin are altered, resulting in the secondary site releasing its hold on trypsin. The structure of several regions previously considered to be rigid is also significantly modified. The inherent flexibility of ecotin allows it to accommodate these mutations and still maintain tight binding through the compromises of the protein-protein interfaces in the ecotin-trypsin tetramer. A comparison with two recently described ecotin-like genes from other bacteria suggests that these structural and functional features are conserved in otherwise distant bacterial lineages.
 ==About this Structure==
-EZU is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] and [http://en.wikipedia.org/wiki/Rattus_norvegicus Rattus norvegicus] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Trypsin Trypsin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.4 3.4.21.4] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1EZU OCA].
+EZU is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] and [http://en.wikipedia.org/wiki/Rattus_norvegicus Rattus norvegicus] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Trypsin Trypsin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.4 3.4.21.4] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EZU OCA].
 ==Reference==
@@ Line 15: / Line 15: @@
 [[Category: Rattus norvegicus]]
 [[Category: Trypsin]]
-[[Category: Craik, C.S.]]
+[[Category: Craik, C S.]]
-[[Category: Fletterick, R.J.]]
+[[Category: Fletterick, R J.]]
-[[Category: Gillmor, S.A.]]
+[[Category: Gillmor, S A.]]
 [[Category: Takeuchi, T.]]
-[[Category: Yang, S.Q.]]
+[[Category: Yang, S Q.]]
 [[Category: CA]]
 [[Category: macromolecular complex]]
@@ Line 25: / Line 25: @@
 [[Category: protein-protein interactions]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 14:28:40 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:33:18 2008''