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Ecotin: a serine protease inhibitor with two distinct and interacting binding sites.
Ecotin: a serine protease inhibitor with two distinct and interacting binding sites.
- Source :
-
Journal of molecular biology [J Mol Biol] 1998 Jun 19; Vol. 279 (4), pp. 945-57. - Publication Year :
- 1998
-
Abstract
- The interaction between ecotin and target proteases with trypsin-like specificity has been systematically dissected to understand the structural basis of ecotin's broad inhibitory specificity and the role of the secondary binding site. Site-directed and region-specific mutagenesis were preformed at ecotin's primary site P1 residue (84), the C-terminal dimer interface (133 to 142), and two surface loops of the secondary binding site (67 to 70, 108 to 113). Substitutions at the P1 position resulted in less than fivefold difference in the potency of ecotin binding to rat trypsin, suggesting that the extended binding site is important in binding. A ten amino acid C-terminal truncation variant showed threefold weaker self-association but remained a dimer. The interactions of the secondary binding site of ecotin with bovine trypsin, rat trypsin and human urokinase-type plasminogen activator (uPA) were investigated with alanine substitutions in ecotin at Trp67, Gly68, Tyr69, Asp70, Arg108, Asn110, Lys112 and Leu113, which formed contacts between the inhibitor and protease. By combining these mutations at the secondary binding site with mutations in the primary binding site the molecular recognition between ecotin and its target serine proteases was probed. The contrast in the Ki values of the various ecotin variants towards bovine trypsin, rat trypsin and human uPA established the role of ecotin's secondary binding site in recognizing these homologous serine proteases. Ecotin binds to proteases with a chymotrypsin fold through a combination of primary and secondary site surface loops and is amenable to redesign of its potency and specificity for this class of enzymes.<br /> (Copyright 1998 Academic Press Limited.)
- Subjects :
- Animals
Bacterial Proteins genetics
Bacterial Proteins metabolism
Binding Sites
Cattle
Dimerization
Humans
Mutagenesis, Site-Directed
Rats
Serine Proteinase Inhibitors genetics
Serine Proteinase Inhibitors metabolism
Structure-Activity Relationship
Bacterial Proteins chemistry
Escherichia coli metabolism
Escherichia coli Proteins
Periplasmic Proteins
Serine Proteinase Inhibitors chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0022-2836
- Volume :
- 279
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 9642073
- Full Text :
- https://doi.org/10.1006/jmbi.1998.1748