What is Ecotin

Ecotin (eco) is a potent inhibitor of serine proteases that is derived from Escherichia coli. It was originally named for its ability to inhibit trypsin (E. coli trypsin inhibitor), but it is known to interact with and inhibit virtually all characterized tryp-sin-fold serine proteases. It is insensitive to the active site P1 preference of the protease (the amino acid N-terminal to the cleaved or scissile bond1)) and inhibits proteases with specificity towards basic, large hydrophobic, small aliphatic and acidic amino acids [2]. This remarkable breadth of inhibition classifies eco as a fold-specific inhibitor. It forms a unique tetrameric complex consisting of two protease molecules and two inhibitor molecules (the E2P2 complex), binding in a bi-dentate manner with two surface loop regions known as the primary and secondary sites (3) (Fig. 7.1). Eco itself is a 142 amino acid protein that forms a stable

1) Serine protease substrate recognition sites are labeled according to the method of Schecter and Berger [1].

Protein Crystallography in Drug Discovery Edited by R. E. Babine and S.S. Abdel-Meguid

Copyright © 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30678-1

Secondary Binding —~2 Site

Dimerizalitin Interface

Dimerizalitin Interface

Primary Binding Site

Fig. 7.1 The tetramer of eco bound to a serine protease. Visualized as a cartoon of the canonical protease and eco interaction (a), and (b), as two views of the three dimensional solution of D102N trypsin in complex with eco [3]. Each eco molecule has three protein-protein interaction surfaces. The C-terminus forms an anti-parallel ft ribbon to complete the ecotin dimer interface. The 80's and 50's loops form the primary binding site by interacting with the protease at the active site cleft in a substrate-like ft-sheet conformation. The 60's and 100's loops of eco form the secondary binding site by interacting with the C-termi-nal a-helix of the protease. Note that each eco molecule contacts both of the protease molecules. Two eco molecules (black and medium grey) form a pair of interactions each with two protease molecules (light grey). The catalytic triad residues Ser-195, Asp-102 and His-57 are in black ball and stick representation. This figure was made with Molscript [37] and Raster 3D [38].

17 kDa dimer held by an arm-in-arm anti-parallel fi ribbon interaction between the two C-termini. The extensive dimer interface characterizes eco as a domain swapped dimer [4]. The three-dimensional structure of eco is composed entirely of fi strands and has been described as a modified jellyroll structure [3]. Separate segments of the eco chain form two spatially independent binding sites to bind to two protease molecules [3, 5].

The primary site binds in a manner that mimics a protease substrate, forming between three and eight main chain hydrogen bonds in a fi-sheet conformation along the active site cleft. This substrate-like binding defines the substrate binding cleft in seven serine proteases to date. The 80's loop of eco makes an extended interaction with the protease while the 50's loop of eco has two functions. It stabilizes the 80's loop through the formation of hydrogen bonds and a disulfide bridge between Cys-51 and Cys-86, and interacts directly with the protease. Eco is a potent inhibitor due to the primary site loops, but the method of the inhibition is still undefined. It is proposed that the protease is inhibited by excluding water from the active site and preventing deacylation of the acyl-enzyme intermediate, the second step in hydrolysis [6]. The eco 80's loop C-terminal to the cleaved bond may also stay strongly associated with the active site and increase the reverse, ligating reaction [7].

The secondary site of eco binds to the protease over 20 A away from the active site and forms up to 30 van der Waals interactions and up to five additional hydrogen bonds. An additional important source of binding energy and association, the secondary site is composed of the 60's and 100's loops of eco and a hydrophobic patch near the protease residues 91 to 94 and the C-terminal a helix amino acids 236 to 242. This patch and helix separated from the 80's loop accounts for the fold specificity of eco. Each inhibitor molecule forms an interaction with both proteases of the tetramer in a clamp configuration that can be adapted to fit most serine proteases.

Because of the large number of proteases it inhibits, WT (wild type) eco has become useful as a biochemical tool. As a selective inhibitor of serine proteases, eco has been used in cell culture assays to probe enzymatic activity as well as to titrate the percentage of catalytically active molecules of a previously uncharacter-ized serine protease [8]. Immobilized eco has been used for the direct purification of trypsinogen [9]. Finally, eco selectively identified a novel protease implicated in prostate cancer, membrane type serine protease I (MT-SP1) [10].

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  • Gebre
    What is the drug ecotin working for?
    2 years ago

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