What are the serpins?

1. What are the serpins? • It is a family of proteins characterised by a common molecular architecture • Most of the serpins are serine protease inhibitors, but some of them have other functions • Today, more than 500 serpins have been identified in animals, plants, bacteria and viruses

2. Serpin structure • Inhibitory mechanism of serpins • Serpin polymerisation

3. PAI-1 (plasminogen activator inhibitor type 1),the only serpin which spontaneously converts to the latent form • inhibits the plasminogen activators, uPA and tPA • regulates fibrinolysis (dissolving of blood clots) and cell migration • the specificity and stability of PAI-1 is regulated by cofactors such as heparin and vitronectin • lacks cysteine residues (from Sharp et al. 1999) Why PAI-1 spontaneously converts to latent form?

4. Distance measurement using donor-donor energy migration (DDEM) Time

5. Localisation of the RCL in PAI-1 by intramolecular distance measurements Distances determined by the DDEM method (Å) Distances in stable PAI-1 mutant (X-ray ) (Å) Distances measured P1’ - 313 P3 - 313 69 68 55  2 55  2

6. Complex Intact Cleaved Oxidized Preinsertion of the RCL studied by the ability to form intramolecular disulfide bonds Conclusion: formation of disulfide bonds between the cysteines in RCL and cysteines in the A--sheet suggests that the RCL in active PAI-1 can be preinserted.

7. Conclusion In contrast to other serpins, active PAI-1 has RCL located close to the core and preinserted. This may be a reason why PAI-1 spontaneously converts to latent form. P. Hägglöf et al., J. Mol. Biol. 2003.

8. Inhibitory mechanism of serpins • What was known: • serpins form very stable/irreversible complexes with their target proteases • when the complexes were analysed by SDS-PAGE or amino acid sequencing, the serpins were cleaved • Major questions: • Are serpins cleaved in the native complexes or the cleavage is an artifact of the analyses? • How look the serpin/protease complex?

9. Quantification of free N-terminals in native serpin/protease complexes Result: in native serpin-protease complexes the N-terminus of PCF is blocked to the same extent as the other N-termini PCF Conclusion: in the native serpin/protease complex the reactive centre of serpin is cleaved and the protease covalently bound to the serpin M. Wilczynska, et al., J. Biol. Chem. 1995.

10. What is the conformation of serpin/protease complex?

11. Hypothetical conformations of stable serpin/protease complex

12. Distance measurement in thePAI-1/uPA complex Conclusion: the distance data exclude the “docking conformation” of the PAI-1/uPA complexbut does not distinguish between full and partial-insertion models X M. Wilczynska, et al., Nat. Struct. Biol. 1997.

13. Structural analysis of PAI-1/uPA complex by distance measurement and triangulation Distances (Å) Model of the complex P3-266 P3-185 P3-P1’ P3-313 43,6 34,2 60,3 39,2 49,8 52,1 60,3 8,6 52 52 60 <30 Partial-insertion model Full-insertion model Distances measured by DDEM Conclusion: the distances measured are compatible with full-insertion model M. Fa, et al., Struct. Fold. and Des. 2000.

14. Serpin inhibitory mechanism is driven by serpin metastability Serpin inhibition involves reactive center cleavage and full loop insertion, so the covalently linked protease is translocated from the initial docking site to distal end of serpin.

15. Loop-sheet polymerisation of serpins • Wild-type serpins polymerise only under mild denaturing conditions. • Some of natural serpin mutants spontaneously polymerise in vivo. This results in diseases like cirrhosis and emphysemia (polymerisation of 1-antitrypsin), angioedema (polymerisation of C1-inhibitor), and dementia (polymerisation of neuroserpin). • The polymerisation is accompanied by loss of inhibitory activity.

16. What are the molecular determinants of PAI-2 polymerisation? Plasminogen activator inhibitor type 2, PAI-2, the only serpin which polymerises as wild-type protein • PAI-2 exists as: * extracellular glycosylated form * intracellular non-glycosylated form • PAI-2 has the largest CD-loop in the serpin family

17. Comparison between PAI-2 and 1-AT Conclusion: the breach region does not determine the polymerisation ability of PAI-2 Breach region M. Wilczynska et al., Febs Lett. 2003

18. Polymerisation of native and DTT-reduced PAI-2 Non-denaturing PAGE Native Reduced PAI-2 PAI-2 Conclusion: reduction of PAI-2 makes the protein resistant to polymerisation.

19. Non-denaturing PAGE Identification of a cysteine which is important for polymerisation ability of PAI-2 Conclusion: Substitution of C79 or C161 to serine makes PAI-2 resistant to polymerisation.

20. Analysis of trypsin-degraded wt PAI-2 by Maldi-tof mass spectrometry Cysteines 79 and 161 form disulfide bond

21. 2 Polymerisation of PAI-2 mutant with two cysteines only (C79 and C161) under different redox conditions Conclusions: • The polymerogenic form PAI-2 is stabilised by theC79/C161 disulfide bond. • The polymerogenic and stable monomeric forms of PAI-2 are interconvertible. ? Oxidation Polymerogenic form Stable monomerogenic form

22. Triangulation of the C79 in stable monomeric PAI-2 by intramolecular distance measurements using DDEM Conclusion: Stable monomeric form of PAI-2 has the CD-loop folded on a side of the molecule

23. Is the translocation of CD-loop in PAI-2 linked to conformational changes in the A-β-sheet of the inhibitor?

24. Annealing of synthetic RCL-peptide into wt PAI-2 and its mutants to compare the opening of the A-b-sheet Conclusion: the A--sheet of PAI-2 is more open in the polymerogenic form than in the stable monomeric form of the inhibitor.

25. Conclusion Reduction Oxidation 50 Å Polymerogenic form of PAI-2 Stable monomeric form of PAI-2 Conversion of PAI-2 from the polymerogenic form to the stable monomeric form is accompanied by closing of the b-sheet A and by translocation of the CD-loop from the bottom to the side of the molecule.

26. Do the polymerogenic and stable monomeric forms of PAI-2 exist in nature? Polymerisation of PAI-2 from the cytosol (wt PAI-2) and from the secretory pathway (SP-PAI-2) of CHO cells • Conclusions: • in the cytosol, PAI-2 exists mainly in the stable monomeric form • in secretory pathway, PAI-2 is in the polymerogenic form.

27. Interaction of PAI-2 with vitronectin Conclusion: PAI-2 can form disulfide-bond to vitronectinviathe C79 in CD-loop

28. Reduction polymerisation Oxidation 50 Å Polymerogenic form of PAI-2 Stable monomeric form of PAI-2 Conclusions • PAI-2 is a unique serpin with two mobile loops: the RCL and the CD-loop • The CD-loop of PAI-2 is a redox-sensitive molecular switch that regulates conversion between the polymerogenic andthe stable monomeric forms of PAI-2. • Polymerisation of PAI-2 in vivo may be regulated by redox status of the cell. • Disulfide-binding of vitronectin to the C79 in the CD-loop of PAI-2 may stabilise the active PAI-2 in extracellular compartments. M. Wilczynska et al., EMBO J. 2003; S. Lobov et al., J. Mol. Biol. 2004.