1. CZ5211 Topics in Computational Biology Lecture 6: Biological Pathways I: Molecular Interactions Prof. Chen Yu Zong Tel: 6874-6877 Email: yzchen@cz3.nus.edu.sg http://xin.cz3.nus.edu.sg Room 07-24, level 7, SOC1, NUS yzchen@cz3.nus.edu.sg http://xin.cz3.nus.edu.sgyzchen@cz3.nus.edu.sg http://xin.cz3.nus.edu.sg

2. 2 Biomolecular Interaction: Enzyme + Substrate E + S ==> E + P This is a generalization of how a biochemist might represent the function of enzymes.

3. 3 Biomolecular Interaction: Enzyme + Substrate E + S ==> E + P kinase-ATP complex + inactive-enzyme ==> Kinase + ADP + active enzyme K ATPADP P Here is an example of the generalization represented by two different ways.

4. 4 Biomolecular Interaction: Enzyme + Substrate This is another representation. Kinase-ATP complex Active enzyme inactive enzyme ADP

5. 5 Biomolecular Interaction This is a generalization of the representation. AB CDEF …

6. 6 Biomolecular Function A biomolecule’s function can be defined by the things that it interacts with and the new (or altered) molecules that result from that interaction. AB CDEF …

7. 7 Biomolecular Function This representation makes it easy to focus on the interaction part. AB CDEn …

8. 8 A Simple BIND Record The minimal BIND record has 9 pieces of information. AB 1. Short label for A2. Short label for B 3. Molecule type for A4. Molecule type for B 5. Database reference for A6. Database reference for B 7. Where A comes from8. Where B comes from 9. Publication reference

9. 9 An Example BIND Record You can view this record in BIND AB 1. INAD2. TRP 3. Protein4. Protein 5. GenBank GI 36416156. GenBank GI 7301861 7. GenBank Taxonomy ID 72278. GenBank Taxonomy ID 7227 9. PubMed ID 8630257

10. 10 BIND Stores Molecular Interaction Data

11. 11 BIND Stores Molecular Interaction Data

12. 12 BIND Records are Based on Observations All BIND records will have a publication reference and most will specifically list a method(s) used to demonstrate the interaction. AB 1. Short label for A2. Short label for B 3. Molecule type for A4. Molecule type for B 5. Database reference for A6. Database reference for B 7. Where A comes from8. Where B comes from 9. Publication reference

13. 13 Methods for Detecting Interactions. A great deal of interaction data in BIND originates from high-throughput experiments designed to detect interactions between proteins. The most common methods are: –Two-hybrid assay –Affinity purification

14. 14 Experimental Evidence of Interaction in BIND Remaining 1%

15. 15 Experimental Method: Two-Hybrid Assay 1. 2. 3. 4.

16. 16 Experimental Method: Two-Hybrid Assay

17. 17 Experimental Method: Two-Hybrid Assay

18. 18 Experimental Method: Two-Hybrid Assay

19. 19 Experimental Method: Two-Hybrid Assay

20. 20 Two-Hybrid Assay 1. 2. 3. 4. A B Fields S. Song O. Nature. 1989 Jul 20;340(6230):245-6. PMID: 2547163 UASG GAL4-DBD SNF1 SNF4 Transcription activation domain Allows growth on galactose GAL1

21. 21 Some Two-Hybrid Caveats 1. 2. 3. 4. A Does the DBD-fusion have activity by itself?

22. 22 Some Two-Hybrid Caveats 1. 2. 3. 4. A B Is the ‘interaction’ mediated by some other protein? C

23. 23 Some Two-Hybrid Questions 1. 2. 3. 4. A B Are the proteins expressed? Are they over-expressed? Are they in-frame? Are the interacting domains defined? Was the observation reproducible? Was the strength of interaction significant? Was another method used to back-up the conclusion? Are the two proteins from the same compartment?

24. 24 Some Two-Hybrid Caveats 1. 2. 3. 4. B A Is the ‘interaction’ bi-directional?

25. 25 Experimental Method: Affinity Purification A Protein of interest Tag modification (e.g. HA/GST/His) This molecule will bind the ‘tag’.

26. 26 Affinity Purification A The cell

27. 27 B Affinity Purification A The cell Naturally binding protein Lots of other untagged proteins

28. 28 B Affinity Purification A Ruptured membranes Cell extract

29. 29 B Affinity Purification A Untagged proteins go through fastest (flow-through)

30. 30 B Affinity Purification A Tagged complexes are slower and come out later (eluate)

31. 31 B Some Questions about Affinity Purification A Is the bait protein expressed and in frame? Is the bait protein observed? Is the bait protein over-expressed? Are the interacting domains defined? Was the observation reproducible? Was the interactor found in the background? Was the strength of interaction significant? Was the interaction saturable? Was the interactor stoichiometric with the bait protein? Was another method used to back-up the conclusion? Was tandem-affinity purification (TAP) used? Was the interaction shown using an extract or a purified protein? Is the inverse interaction observable? Are the two proteins from the same compartment? Are the two proteins known to be involved in the same process? Is the interactor likely to be physiologically significant?

32. 32 B Some Affinity Purification Caveats A First and most importantly, this is only a representation of the observation. You can only tell what proteins are in the eluate; you can’t tell how they are connected to one another. If there is only one other protein present (B), then its likely that A and B are directly interacting. But, what if I told you that two other proteins (B and C) were present along with A…. B A C

33. 33 B Complexes with Unknown Binding Topology A Which of these models is correct? The complex described by this experimental result is said to have an Unknown Topology. CB A CB A C

34. 34 B Complexes with Unknown Stoichiometry A Here’s another possibility? The complex described by this experimental result is also said to have Unknown Stoichiometry. C A

35. 35 High Throughput Data in BIND Affinity purification: Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry (2002). PMID: 11805837 Two-hybrid: A protein interaction map of Drosophila Melanogaster (2003). PMID: 14605208 Two-hybrid and Affinity purification: A map of the interactome network of the metazoan C. Elegans (2004). PMID: 14704431 Data from these examples can be retrieved from BIND using a PMID search.

36. 36 B How complex Data are Stored in BIND A ? ? C ? Three interaction records.

37. 37 B How Complex Data are Stored in BIND A ? ? C ? A complex record in BIND is simply a collection of interaction records.

38. 38 B Alternate Representations. A ? ? C ? The matrix model (a clique). B A C

39. 39 B Alternate Representations. A ? ? C ? The spoke model. Which model to use? B A C

40. 40 Spoke and Matrix Models Vrp1 (bait), Las17, Rad51, Sla1, Tfp1, Ypt7 Spoke Matrix Possible Actual Topology Bader&Hogue Nature Biotech. 2002 Oct 20(10):991-7 Simple model Intuitive, more accurate, but can misrepresent Theoretical max. no. of interactions, but many FPs

41. 41 A view on real data…matrix model 6 redox enzymes 7 redox enzymes Old yellow enzyme Function?

42. Interaction Kinetics E + S ==> E + P kinase-ATP complex + inactive-enzyme ==> Kinase + ADP + active enzyme K ATPADP P

43. 43 Reversibility of Chemical Reactions: Equilibrium Chemical reactions are reversible Under certain conditions (concentration, temperature) both reactants and products exist together in equilibrium state H 2  2H

44. 44 Reaction Rates Net reaction rate = forward rate – reverse rate In equilibrium: Net reaction rate = 0 When reactants “just” brought together: Far from equilibrium, focus only on forward rate But, same arguments apply to the reverse rate

45. 45 The Differential Rate Law How does the rate of the reaction depend on concentration? E.g. 3A + 2B  C + D rate = k [A] m [B] n (Specific reaction) rate constant Order of reaction with respect to A Order of reaction with respect to B m+n: Overall order of the reaction

46. 46 Rate Constants and Reaction Orders Each reaction is characterized by its own rate constant, depending on the nature of the reactants and the temperature In general, the order with respect to each reagent must be found experimentally (not necessarily equal to stoichiometric coefficient)

47. 47 Elementary Processes and Rate Laws Reaction mechanism: The collection of elementary processes by which an overall reaction occurs The order of an elementary process is predictable UnimolecularA*  BK + [A] First order Bimolecular A + B  C + D K + [A] [B] Second order Trimolecular A + B + C  D + E K + [A] [B] [C] Third order

48. 48 Elementary Processes and Rate Laws Reaction mechanism: The collection of elementary processes by which an overall reaction occurs The order of an elementary process is predictable UnimolecularA*  BK + [A] – K - [B] First order Bimolecular A + B   C + D K + [A] [B] – K - [C] [D] Second order Trimolecular A + B + C   D + E K + [A] [B] [C] – K - [D] [E] Third order