Serine proteases are involved in numerous biological processes, including apoptosis, blood coagulation, viral maturation, and cancer. Highly specific protease inhibitors can be powerful tools for studying proteases and elucidating their in vivo roles. Ecotin, a macromolecular serine protease inhibitor found in the periplasm of E. coli, offers a unique platform for protein engineering. In contrast to most protease inhibitors, ecotin shows very broad specificity and also binds as a dimer to target proteases, forming a tetrameric complex involving ecotin-protease recognition both at the active site and at a distal secondary site. Alanine shaving and phage display experiments on several proteases have indicated that the importance of each of ecotin's two binding sites differs with the specific protease and have also shown that ecotin can be remodeled to make a more potent inhibitor. The blood coagulation cascade involves multiple homologous serine proteases. We intend to create potent and specific inhibitors of one or more of these proteases to better understand this complex process and as a possible therapeutic agent against thrombosis. Utilizing known crystal structures, we are using use computer graphics to model the interaction between ecotin and target proteases. Structure-guided loop-trimming and specificity tuning are used to convert ecotin into a potent inhibitor of these serine proteases. Crystal structures of these ecotin variants complexed with their target proteases will be analyzed to understand the mechanism of protease inhibition. By studying the fundamental structural interactions between ecotin and proteases, we hope to be able to create specific inhibitors for biologically important target proteases.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001081-23
Application #
6347846
Study Section
Project Start
2000-07-01
Project End
2001-06-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
23
Fiscal Year
2000
Total Cost
$136
Indirect Cost
Name
University of California San Francisco
Department
Type
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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