1. By what mechanisms are calcium signals read and translated into biochemical response? What is the structural basis for the function of the proteins involved? How is the specificity of different signalling pathways generated? Calcium Signal Transduction

2. Now we understand how the calcium signal is read and transduced into biochemical response The next step is to examine how the diversity in the functions of EF-hand proteins is achieved.

3. EF-hand Protein Primary Structure Calcium Coordination 7 oxygen atoms from: 3 mono-dentate side chains 1 backbone carbonyl 1 water H-bonded to side chain 1 bi-dentate side chain Specific geometry: pentagonal bi-pyrimid 1 3 5 7 9 12

4. S100 Proteins are Unique CaBPs  S100 proteins have a unique dimeric structure Potts. et al., 1995 2 slides

5. Dimer is the Basic Structural Unit  Interdigitated side chains  A single contiguous hydrophobic core 6 slides

6. All S100 Proteins Have The Same Basic Architecture

7. S100 Protein Mechanism is Unique  S100 proteins have a unique dimeric structure  The mode of signal transduction must be distinct from calmodulin Smaller  changes in conformationSmaller  changes in conformation Target interactions are differentTarget interactions are different Potts. et al., 1995

8. Ca 2+ Target The Calmodulin Paradigm

9. Activation of Typical Ca 2+ Sensors Accessible Hydrophobic Surface Calmodulin N-terminal Domain

10. S100 Response to Ca 2+ Binding is Small Significant change only in Helix III Significant change only in Helix III

11. Ca 2+ -induced Conformational Change S100s Are Different From Calmodulin Ca 2+ Sensor S100B CaM-N S100 protein response is much smaller than typical Ca 2+ sensors S100 protein response is much smaller than typical Ca 2+ sensors

12. S100 Proteins Bind Helical Targets Shift in Helix III exposes binding site Shift in Helix III exposes binding site S100B/p53

13. S100s Must Bind Targets Differently Calcium-dependent regulation of targets: kinases, kinase substrates, receptors (RAGE), fatty acids (arachidonic acid!) Calcium-dependent regulation of targets: kinases, kinase substrates, receptors (RAGE), fatty acids (arachidonic acid!) Calmodulin/MLC Kinase S100B/p53 MLCK p53

14. CONCLUSION S100 proteins are structurally (and functionally) different from classical Ca 2+ sensors -BUT- They are very similar to each other!

15. All S100 Proteins Have The Same Basic Architecture S100A8 S100A9

16. Similarity Extends Beyond Architecture Calbindin D 9k !! A7 A8 A12 A6 B

17. A Bonus: Accurate Homology Modeling S100A4 Ca 2+ -free Ca 2+ -bound  We now know the basic structure of every S100 protein!! So why don’t we know all of the answers?…it’s all about the details!

18. If they are so similar, what provides the functional specificity of S100 proteins?

19. Functional Specificity: Calmodulin Differences in the Binding Sites for Different Proteins Extremely similar structures, but subtle details different Extremely similar structures, but subtle details different calmodulin caltractin

20. Functional Specificity: Calmodulin Differences in the Binding Sites for Different Proteins Differences in charge and shape of the binding surface Differences in charge and shape of the binding surface extra pocket opposite charge extra cleft CaltractinCalmodulin

21. Functional Specificity: S100 Proteins Different Binding Sites for Different Proteins Differences in size and shape of the binding sites Differences in size and shape of the binding sites S100A6 S100B

22. Ca 2+ loaded S100A6 Ca 2+ loaded S100B Apo S100A6 Apo S100B Differences in  hydrophobic surface induced by Ca 2+ binding Differences in  hydrophobic surface induced by Ca 2+ binding Differences in Hydrophobic Surface

23. Differences in Electrostatic Surface S100B-P53 S100A11-Annexin-I S100A11-Annexin-I Complemented by the properties of the target Complemented by the properties of the target

24. Functional Specificity Different Binding Modes for Different Proteins

25. S100A10/annexin II S100A11/annexin I S100A9/Chaps S100B/p53

26. Functional Specificity A New Concept!! S100B-Ndr S100B-p53 Different binding modes for the same Different binding modes for the same S100 protein with different targets!!

27. Functional Specificity Diversity in Quaternary Structure? Dimer Tetramer S100A12 S100A9

28. Functional Specificity Diversity in Quaternary Structure? Dimer Hexamer

29. Dimer Octamer Tetramer Hexamer

30. Summary of Structural Concepts The S100 architecture is unique The S100 architecture is unique Mode of action differs from the CaM paradigm Mode of action differs from the CaM paradigm Structures (apo, Ca 2+ -bound) are very similar Structures (apo, Ca 2+ -bound) are very similar What provides functional specificity? What provides functional specificity?

31. Factors Providing Functional Specificity  Sequence variability of residues at the surface alters the character of binding sites  The complementarity of the binding surface and target leads to different binding modes  Different modes for different proteins  Multiple modes for each protein?  Oligomerization may effect the presentation of binding surfaces to targets

32. Extending Knowledge of Target Interactions

33. S100A8 and S100A9

34. Structures of Both Homodimers S100A8 S100A9 C-terminal tail of S100A9 is missing C-terminal tail of S100A9 is missing Only homology models of heterodimer Only homology models of heterodimer

35. Analysis of S100A8/S100A9 Interactions with Cellular Targets

36. S100A8/A9-Arachidonic Acid Titration Ca 2+ + AA apo Ca 2+ Ca 2+ -bound S100A8/S100A9 binds arachidonic acid Ca 2+ -bound S100A8/S100A9 binds arachidonic acid

37. NMR Analysis of Binding of Arachidonic Acid by S100A8/A9 15 N - C  - CO - - - 15 N - C  H RR H 15 N S100A9 15 N S100A9 + AA

38. NMR Analysis of Binding of Arachidonic Acid by S100A8/A9  Differential effects on protein signals  Discrete binding site for ligand  Still seeking to optimize

39. RAGE Receptor for Advanced Glycations Endproducts V C1 C2 Cell Membrane Variable (V) and Constant (C) IGG domains EN-RAGE receptor ligand: an S100 protein!EN-RAGE receptor ligand: an S100 protein!

40. Limited Proteolysis to Find Structural Domains of RAGE Three putative stable domains: V, C1 and C2 Three putative stable domains: V, C1 and C2 Intact S-RAGE Primary fragments

41. Protection in the Presence of Targets - S-RAGE + S-RAGE The binding of S-RAGE stabilizes S100A8/A9 The binding of S-RAGE stabilizes S100A8/A9

42. Affinity Chromatography Proteolysis Mass Spec Identification EluteWashRAGE ImmobilizedS100

43. S100/RAGE Affinity Chromatography St FT W1 W2 Elution A9 A8 RAGE domain * S-RAGE * Retention of intact S-RAGE and stable domainRetention of intact S-RAGE and stable domain Not pure!!

44. S100/RAGE Affinity Chromatography RAGEdomains S-RAGE Pure Partial Full Affin.. Pure Partial Full Affin.. Digestion Digestion Purified protein gives same result! Purified protein gives same result!

45. Titration of S100A8/A9 With S-RAGE  Differential effects on protein signals  Discrete binding site for S-RAGE