Adhesion P. gingivalis protease

1. Adhesion P. gingivalis protease Secretion signal peptidases Immune Response T-cell protease Development snake Blood pressure regulation renin Digestion trypsin Coagulation thrombin Complement Fixation CI protease Cell fusion hemaglutinase Tumor Invasion collagenase Reproduction and Fertilization acronase Pain Sensing kallikrein Fibrinolysis tissue plasminogen actvator Animal Virus Replication HIV protease Hormone Processing Kex 2 6 Broad Categories FunctionProtease Nutrition trypsin, subtilisin, a-lytic protease Invasion matrix metallo proteases Evasion IgA protease Adhesion P. gingivalis protease Processing signal peptidase, viral proteases, proteosome Signaling caspases, granzymes

2. What do proteases do? H O H + H2O + +3HN C C N C COO- R1 H R2 H +3HN C COO- R1 H +3HN C COO- R2 DGo for the rxn is -2kcal/mol But… Catalyzed rxn (chymotrypsin) at neutral pH, 37°C: 100/sec Uncatalyzed rxn at neutral pH, 37°C: 1 X 10-10 /sec Conditions for chemically catalyzed reaction: 24hrs. @ 6M HCl, 110°C Koshland, D. (1996) J. Cell. Comp. Phys. Suppl. 1 43:217.

3. endopeptidase exopeptidase Two types of cleavages Same rxn, Four mechanisms Named for residue/group in active site of enzyme essential for most effective catalysis Serine -OH Cysteine/Thiol -SH Acid/Aspartic COO- Metallo Zn2+

4. Mechanistic Sets of Proteases set feature inhibitor examples function Serine protease active site serine fluorophosphates trypsin digestion H57, D102, S195 thrombin blood coagulation plasmin lysis of blood clots coccoonase mechanical subtilisin digestion acrosin sperm penetration Cysteine protease active site cysteine iodoacetate papain digestion C25, H159, N175 strept. proteinase digestion cathepsin B intracell. digestion Acid protease acidic pH optimum diazoketones pepsin digestion D32, D215 chymosin milk coagulation Metalloproteases Zn2+, E270 o-phenanthroline carboxypeptidase digestion Zn2+, Ca2+ o-phenanthroline thermolysin digestion E143, H231

5. Serine Protease Mechanism – The players Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

6. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

7. Serine Protease Mechanism – Oxyanion Hole Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

8. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

9. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

10. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

11. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

12. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

13. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

14. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

15. Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

16. Serine inhibitors CH3 O O S NH CH C CH2 Cl O CH2 Peptide bond mimic Chloro-methyl ketone [CMK] TPCK (L-1-Chloro-3-[4-tosylamido]-4-phenyl-2-butanone)

17. Serine inhibitors CH3 F CH3 CH O P O CH O CH3 CH3 DFP Diisopropyl fluorophosphate

18. Divergent vs. Convergent Evolution Catalytic Triad Conserved Trypsin Elastase Subtilisin Same Fold

19. Serpins Serine protease inhibitors Irreversible Disruption of 3º structure

20. Ecotin Serine Protease Inhibitor Unknown function Dimeric 1° and 2° binding sites Cleaved

21. Cysteine protease mechanism 159 25 S N: N H H O HN 159 25 P1 + H N N S H O- O H P1 159 25 + N H N S H O- 159 HN 25 P1 N: N S H O NH2 P1 Michaelis Complex Tetrahedral intermediate I NH2 Tetrahedral intermediate II H2O Acyl Intermediate

22. Cysteine protease mechanism 159 25 S N: N H H O HN 159 25 P1 + H N N S H O- O H P1 159 25 + N H N S H O- 159 HN 25 P1 N: N S H O NH2 P1 Michaelis Complex Tetrahedral intermediate I Covalent Intermediate No Asp102 equivalent NH2 Tetrahedral intermediate II H2O Acyl Intermediate

23. Cysteine protease inhibitors 159 25 S N: N H H I O CH2 C OH Iodoacetic acid E-64 (2S,3S)-3-(N-(1S)-1-[N-(4guanidinobutyl)carbamoyl]3methylbutyl)carbamoyl) oxirane-2-carboxylic acid

24. Cystatin Superfamily Cysteine protease inhibitors Non-canonical binding

25. Acid protease mechanism P1’ P1 N O H P1 P1’ H N O O O H -O - O H H O O O Asp25 - P1 H H O O O P1’ P1 P1’ H H O N N H O- H H O H O O O O O O- O - - H H Michaelis complex H O O Asp25’ Asp25 Asp25’ Tetrahedral intermediate Asp25 Asp25’ Asp25 Asp25’

26. Acid protease mechanism P1 P1’ P1’ P1 H N N O O O H H O O H H O H O O O -O O - - P1 H H O O O P1’ P1 P1’ H H O N N H O- H H O H O O O O O O- O - - H H Michaelis complex Asp25 Asp25’ Asp25 Asp25’ No covalent intermediate Activated water Tetrahedral intermediate Asp25 Asp25’ Asp25 Asp25’

27. Acid protease inhibitors Indinavir, Roche

28. CH3 O HN HIV Protease Substrate NHR’ RHN N O O OH Reiling, K. K. et al. Biochemistry (2002) 41:4582-94.

29. Movie of Multi-drug resistant HIV Models: www.ucsf.edu Click on A-Z listings Under C find Craik, Charles Within the Craik website there is section entitled movies Enjoy!

30. Pepsin HIV Protease

31. Metallo protease mechanism Glu H - His O O His Zn2+ His Glu O H Glu His H O Zn2+ His O Zn2+ H O O O N H -O -O His P1 P1’ - - His His Glu His His Glu Zn2+ - O O H Zn2+ O O O P1 O- H P1 His N His Glu P1’ Zn2+ O O P1 H H N P1’

32. Metallo protease mechanism Glu H - His O O His Zn2+ His Glu O H Glu His H O Zn2+ His O Zn2+ H O O O N H -O -O His P1 P1’ - - His His Glu His His Glu Zn2+ - O O H Zn2+ O O O P1 O- H P1 His N His Glu P1’ Zn2+ O O P1 H H N P1’ No covalent intermediate Activated water

33. A Zn2+ H H O H O H O H2N NH C C N C C N C C C NH Arg R2 H R1 H R1 O H2N A Zn2+ H H O H O H2N S CH2 C C N C C C NH Arg CH3 O H2N H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-His-Leu-Co2H Proangiotensin H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-Co2H Angiotensin - + carboxy-di-peptidase active site + - Captopril

34. Thermolysin Carboxypeptidase A

35. Synopsis of Protease Mechanisms Serine Ser-His Asp Catalytic Triad covalent intermediate Cysteine Cys-His covalent intermediate Acid Asp-Asp Activated water no covalent intermediate Metallo Zn2+ or equivalent-Glu Activated Water no covalent intermediate

36. How Proteases Order Off the Menu OH CH3 O O HN HN NH NH NH O O OH NH3+ P2 P1 P1’ P2’ Peptide Scissile Bond Subsite of Protease S2 S1 S1’ S2’

37. Substrate Selection within One Tertiary Fold

38. Methods to Determine Specificity 1> Synthesis of short peptides [15 to 20a.a.], check for cleavage with PAGE 2> Phage display of short peptides 3> Positional scanning synthetic combinatorial libraries [PS-SCL]

39. X O O X HN HN NH NH X X O O HN O 7-amino-4-methyl coumarin R N D E Q G H I L A Ac-XXXO-AMC K F P S T W Y V m R N D E Q G H I L A Ac-XXOX-AMC K F P S T W Y V m R N D E Q G H I L A Ac-XOXX-AMC K F P S T W Y V m R N D E Q G H I L A Ac-OXXX-AMC K F P S T W Y V m Harris J. L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. PNAS (2000) 97:7754-9.

40. P2 O O P4 HN HN NH NH P1 P3 O O

41. Regulation of Proteases – A Few Examples Trypsinogen 1 1 16 15 Trypsin 16 Zymogens Pro-peptide that must be cleaved before protease becomes fully active Enteropeptidase Zymogen form has distorted oxyanion hole and substrate binding pocket Compartmentalization Macromolecular Inhibitors Host and non-host

42. Cytotoxic Lymphocytes Molecular Biology of the Cell, Garland

43. Cytotoxic T Lymphocyte Apoptotic Pathway Cytotoxic T lymphocyte Granzymes Perforin Ca2+ Ca2+ Ca2+ 3 Fas Ca2+ GrnA GrnB MPR? DD FADD cleave pro-caspases serpins Nuclease? DED Mito. apoptosis Single stranded breaks in DNA Bcl-2 aggregrates pro-caspase 8, intermolecular cleavage to caspase 8, activation of effector caspases [3, 6, 7], apoptosis nucleus

44. Granzymes: Lymphocyte Serine Proteases Name Activity Predicted P1 MW cleavage site A Trypsin-like R/K 60 (Dimer) BAsp-ase D/E 35 C Unknown N/S 27 D Unknown F/L 35-50 E Unknown F/L 35-45 F Unknown F/L 35-40 G Unknown F/L H Chymase F I Unknown J Unknown K Trypsin-like 30 M Met-ase M/L/nor-L 30

45. Granzyme Structure Waugh et al. (2000) Nat. Struct. Biol. 7:762-765

46. Granzyme A, Proposed Dimeric Structure

47. Substrate Sequence P4 P3 P2 P1 FLUOROGENIC LIBRARIES V/I G/A/S N R PIL-1b D A P V R S L N C T THROMBIN RECEPTOR T L D P R S F L L R HISTONE H1 K L G L K S L V S K HISTONE H2b A P A P K K G S K K SET Q T Q N K A S R K R LAMIN B V T V S R A S S S R Granzyme A: Substrate Specificity and Macromolecule Substrates

48. Chasing the Crystals

49. mOD/min @ 405nm 0 0.05 5 50 [Inhibitor], mM Macromolecular Inhibition of Granzyme A Control mM84R Eco dM84R Eco Tryp. Inh.

50. Potential Effects of Oligomer on Macromolecular Inhibitors grnA