Bioinformatique et Biologie Structurale I/ – Principes et techniques

1. Bioinformatique et Biologie Structurale I/ – Principes et techniques A/ L’information structurale B/ Les différentes techniques de détermination de structure C/ Les nouveaux challenges de la biologie structurale II/ – Application à l’étude d’enzymes d’intérêt médical A/ Un bref aperçu de ce que l’on appelle « Drug design » B/Recherche d’inhibiteurs d’aminopeptidases de Streptocoques C/ Relations structure-fonction d’hélicases impliquées dans les cancers

2. X-PDAP activity exopeptidase Selectivity : Pro ; ALA 10%AS ; GLY 1 % AS Two families : S9B and S15 for the same enzyme activity S9B :DPP-IV [eukaryotic and prokaryotic] membrane-bound and soluble forms S15 :PepX [prokaryotic] cytoplasmic

3. X-PDAP activity • Proline specific proteases : a rare group • Many biologically active peptides contain an evolutionary conservedproline residue as a proteolytic processing regulatory element • Proline-specific proteases : important 'check-points' control • Importance in some disease to inhibit such proteases • X-PDAP: S9b and S15 family evolutionarily distant enzymes DPP-IV and PepX • Importance of the enzyme activity in prokaryotics • Proteases and peptidases have been identified as critical virulence factors in numerous microbial pathogens ; may act on a variety of host proteins including serum and tissue components thus contributing to neutralization of the immune defense system and tissue invasion and destruction. • DPP-IV is involved in various mammalian regulation processes and in serious human diseases (Diabetes type II,…)

4. The signature of the X-PDAP specificity in SC Clan enzymes • Structure / function relationships in X-PDAP enzymes • What makes the enzymes so specific? • - Clans, families of proteases and X-PDAP activity • Structure of PepX, X-PDAP from Lactococcus lactis • Comparison of bacterial and human X-PDAP structures • Insights for drug design • Conclusion

5. SC Clan • Almost all enzymes are specialized in cleavages involving a proline residue • Oligopeptidases [endo, release peptides] • Iminopeptidases [exo, release PRO] • Carboxypeptidases [release peptides] • X-prolyl dipeptidyl aminopeptidases [exo, releases X-PRO] • endo detected in the case of DDP-IV

6. http://merops.sanger.ac.uk/

7. S15 family - Alignments - + legend figure 1 HERE!

8. Structure Resolution of PepX from Lactoccocus lactis 10 % PEG 4000, 150mM NaCl Ph 5.2 MES NaOH, 18°C

9. C-terminal helical lasso catalytic N-terminal PepX prototype de la famille S15 - 4 domaines - a/b hydrolase fold - éléments remarquables (peptide lasso, Boucle C-ter, …) 85 Å 25 Å Rigolet, P. et al. (2002). Structure10, 1383-1394

10. Enzymes families of SC Clan and related structures Catalytic domain(a/b hydrolase fold) in green, N-ter domain in red, C-ter domain in blue and helical domain in orange

11. Comparison of sequence and structures of SC Clan enzymes SPAP [317 residues] S33 family CBPY [416 residues] S10 family POP [710 residues] S9A family DPP-IV [726 residues] S9B family CBPY [416 residues] S10 family 228 CA (3.03 Å) 17.5 % POP [710 residues] S9A family 180 CA (3.17 Å) 15.3 % 154 CA (3.47 Å) 17.6 % DPP-IV [726 residues] S9B family 207 CA (3.11 Å) 10.8 % 189 CA (2.95 Å) 11.9 % 451 CA (3.50 Å) 19.8 % PepX [763 residues] S15 family 175 CA (3.02 Å) 16.4 % 184 CA (3.30 Å) 12.3 % 207 CA (2.63 Å) 16.8 % 201 CA (3.15 Å) 17.8 %

12. Structure comparisons between PepX and : Cocaine Esterase (COCE,) 1JU3 , 565 residues 40% Specific to PepX Prolyl Iminopeptidase (XCPIP, Xant. campestris) 1JU3 , 313 residues 66% Specific to PepX Prolyl Oligopeptidase (POP, porcine muscle) 1JU3 , 710 residues 64% Specific to PepX

13. Structure-based sequence aligment Only 4 conserved sequences can notably be distinguished between the two sequences of PepX and DPP-IV: - sequence NxxxAxxGxSYxG around the active serine ; - sequenceLxxHGxxDxNVxxxxQxxxxxKAL around the activeaspartic acid ; - short sequence HxxxxxS after the activehistidine ; - sequence AxAxxSxWxxY before the Pos1 subsite of the X-PDAP signature.

14. PepX Dimeric structure • Important for activity • Globular shape • Involve principally N-ter and helical domains • Canal acces to catalytic residues • The two active sites are far away from each other and independently accessible to the substrate • Hydrophylic interface; labile contacts

15. DPP-IV Dimeric structure DPP-IV Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U., Kiefersauer, R., Huber, R., Bode, W., Demuth, H.U. and Brandstetter H. (2003). Proc. Natl. Acad. Sci. USA 100, 5063-5068.

16. PepX Electrostatic properties Acidic surface (also seen for other proteases of Lact. lactis ; adaptation to a particular cellular environment)

17. DPP-IV Electrostatic properties DPP-IV Rasmussen HB, Branner S, Wiberg FC, Wagtmann N. (2003). Nature Struct. Biol.10, 19-25.

18. Differences between the two enzymes • Nearly the same lenght (PepX 763 and DPP-IV 766) • Different folds (moreover 4 versus 3 domains) • Both dimer but of different quaternary organization • DPP-IV is integrated in the plasmatic membrane whereas PepX is an cytoplasmic enzyme • Substrate selection processes via an N-ter propeller domain whereas via dimeric domain in PepX.

19. Comparison of the specificity sites of PepX and DPP IV Evolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity.

20. Comparison of the specificity sites of PepX and DPP IV Oxyanion hole Evolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity.

21. Comparison of the specificity sites of PepX and DPP IV Signature X-PDAP activity Evolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity.

22. Comparison of the specificity sites of PepX and DPP IV ? Evolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity.

24. Structural signature of the XPDAP activity Equivalent residues in compared enzymes of the clan SC. Rigolet P. et al. . (2005). FEBS J. 272; 2050-2059.

25. Recherche d’inhibiteurs spécifiques • Tester inhibiteurs de DPPIV • (ceux de petite taille) • Exploiter différences : • R125 DPP-IV • L401 PepX / Y666 DPPIV • Structure dePepX avec un inhibiteur deDPP-IV

26. Recherche d’inhibiteurs spécifiques Table 2: Inhibition experiments realized with PepX from Lactococcus lactis.

27. Recherche d’inhibiteurs spécifiques KI = 9 mM Valine-Pyrrolidide Inhibition compétitive IC50 = 30 mM KI = 9 mM Rigolet P., Xi, X.G., Rety, S. and Chich, J.F. (2005). FEBS J. 272; 2050-2059.

28. Recherche d’inhibiteurs spécifiques Table 2: Inhibition experiments realized with PepX from Lactococcus lactis. Table 3: Docking simulations of valine-pyrrolidide in the X-PDAP enzymes.

29. Recherche d’inhibiteurs spécifiques Nouveaux dérivés partant de la valine-pyrrolidide … … Nous les avons synthétisés …. … Nous les testons actuellement ….

30. Drug design: autres stratégies - interface de dimérisation • boucles C-TER - Mutagenèse dirigée : PHE 80

31. Quelle est la fonction des domaines Nter et Cter? • Qu’apportent-ils vraiment à la catalyse ? • Rôle présenté chez DPPIV • Pas de réelle interprétation ni chez aAeH ni chez Coce • Rôle supposé chez PepX, qui possède à la fois un Nter et un Cter • Siège d'une deuxième fonction ?

32. Collaboration avec CHIMISTES de l’ENS-Cachan Equipe de Joane XIE Synthèse organique d'inhibiteurs potentiels Contributions • P. Rigolet INRA – LURE – ENS Cachan • J. F. Chich INRA • M. M. Delage INRA • I. Mechin EMBL