Tobias Kromann-Hansen & Hans Peter Sørensen

1. Allosteric Inhibition of Murine Urokinase-Type Plasminogen Activator The intradomain linker prevents allosteric transition of murine urokinase-type plasminogen activator into an inactive zymogen-like conformation with a blocked active-site Tobias Kromann-Hansen & Hans Peter Sørensen

2. Background • mU1 is a monoclonal antibody selected against murine uPA by the Finsen Laboratory at Rigshospitalet (Lund, Hansen et al. JBC 2008) • mU1 successfully targets uPA dependent processes in vivo Lund, Hansen et al. JBC 2008

3. Activation of pro-uPA • uPA activation occurs through a single peptide bond cleavage at Lys15-Ile16 • The newly formed N-terminus is inserted into a hydrophobic pocket and forms a salt-bridge to Asp194 • This interaction stabilizes the active-site Andreasen, Kjøller et al., Int. J. Cancer, 1997

4. mU1 is not binding directly to the active site- mU1 is an allosteric antibody competitively inhibiting murine uPA - Kromann-Hansen, T., Lund, IK., Zhou, L. Andreasen, PA., Høyer-Hansen, G., Sørensen, HP. In preparation

5. mU1 is not binding directly to the active site- mU1 is an allosteric antibody competitively inhibiting murine uPA -

6. mU1 inhibits the catalytic domain 20-fold more potently than the full length enzyme Kromann-Hansen, T., Lund, IK., Zhou, L. Andreasen, PA., Høyer-Hansen, G., Sørensen, HP. In preparation

7. The linker region has functional interactions to the catalytic domain Kromann-Hansen, T., Lund, IK., Zhou, L. Andreasen, PA., Høyer-Hansen, G., Sørensen, HP. In preparation

8. The linker region has functional interactions to the catalytic domain Kratz , Mackman et al. Chem. Biol., 2000

9. Binding of mU1 to murine uPA exposes the N-terminus Kromann-Hansen, T., Lund, IK., Zhou, L. Andreasen, PA., Høyer-Hansen, G., Sørensen, HP. In preparation

10. The intradomain linker prevents allosteric transition of murine urokinase-type plasminogen activator into an inactive zymogen-like conformation with a blocked active-site