Organ  Tissue  Cell  Molecule  Atoms

1. INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY(ICGEB), NEW DELHI, INDIA Structural and Computational Biology Group

2. Structural Biology Medicine and Biology at Atomic Scale Organ  Tissue  Cell  Molecule  Atoms

3. High Resolution Structural Biology Atomic structure - communication

4. Anti-tumor Activity Duocarmycin SA Evolution: Machine and Control Atomic interactions

5. NER RPA BER RR Biological linguistics

6. Techniques propel discoveries NMR Spectroscopy X-ray Crystallography Computation Atomic maps

7. RPA-A RPA-B Fluorescence Intensity RPA-AB Ratio of T-ag/RPA Experimental Buffet

8. X-ray NMR Diffraction Pattern X-rays • Direct detection of atom positions • Crystals • Indirect detection of H-H distances • In solution 3D Molecular Structures

9. Flavours… • X-ray- highest resolution, automation • NMR- enables solution variations; • direct tap on motions and on weak interactions • Computation- fundamentals of structure, dynamics

10. Structures breathe Need to incorporate motion

11. Challenges… • 3D structures are static • Biological process (recognition, interaction, chemistry) are dynamic • New methods for molecular motions

12. Malaria Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malaria ~40/400 are vectors Anopheles gambiae

13. Plasmodium “…from yeast to man” Trypanosoma Baldauf, Science 300, 1703 (June 2003) Biology of model organisms - relevance to the malaria parasite?

14. Example 1

15. Duffy-binding-like domains from the erythrocytic stage Invasion of erythrocytes by malaria parasites

16. RBC invasion • apical orientation • microneme secretion • junction formation • receptor/ligand interactions • rhoptry discharge Electron micrograph from Aikawa et al (1978) J. Cell Biol. 77:72

17. The Duffy-Binding-Like (DBL) Superfamily Erythrocyte invasion: mediated by Erythrocyte binding protein family SS I II (DBL) III - V VI TM CYT P. vivax / P. knowlesi P. falciparum DBL F1 F2 Cytoadherence: mediated by PfEMP-1 family DBL1 CIDR1 DBL2 DBL3 DBL4 TM Exon 2 P. falciparum

18. P. vivax RBC invasion P. falciparum RBC invasion Sialic acid/GA Duffy antigen Sialic acid/GC unknown unknown Erythrocyte Erythrocyte

19. Overall domain architecture of PkDBLa

20. DARC-PkDBLa engagement Sulfation of tyrosine 41 on DARC increases binding affinity ~1000X Polar Apolar Polar

21. Invasion and evasion Haemagglutinin gp120 PkDBLa Sialic acid binding site CCR5 binding site DARC binding site Antigenic shift/drift by Conformational masking, Just-in-time release? sequence variation glycan shield, mutants

22. P. vivax versus P. falciparum

23. Structural and functional conservation - mechanistic divergence Pk DBL Pf F1+F2 DBLs F1 : F2 = 1.9Å (185 Ca) F1 : Pka-DBL = 1.6Å (195 Ca) F2 : Pka-DBL = 1.8Å (156 Ca)

24. Structural and functional conservation - mechanistic divergence Insertions

25. Structural and functional conservation - mechanistic divergence P. vivax/P. knowlesiP. falciparum Module duplication Monomeric assembly Dimeric assembly Insertions

26. P. vivax Invasion P. falciparum Polymorphic sites Subdomain 3 loop DARC binding site RBC RBC RBC

27. Example 2

28. UIS3 from the pre-erythrocytic stage Proteins that play crucial roles for the parasite

29. Entry and Development – Liver Stages

30. Structural congruence, functional divergence

31. Genetics and structure driving insights into function

32. Genetics and structure driving insights into function

33. Genetics and structure driving insights into function

34. Example 3

35. AUGCCUUA PFG27 SH3 SH3 Gametocytogenesis in P. falciparum Novel fold Unique 3D structure RNA binding SH3 binding PxxP motifs Signaling intermediate

36. Review of binding sites of interest on Pfg27 • Two RNA-binding sites per dimer • Four SH3-binding sites per dimer • A dimer interface Pfg27 monomer RNA-binding site Deep cavity SH3-binding site Dimer interface

37. Docking pattern on Pfg27 Visual analysis of top 200 dockings FlexX GOLD Deep cavity RNA-binding site Dimer interface SH3-binding site Other sites 2045 3030 3020 105 10 - (values in percent)

38. Docking at RNA-binding site deeper surface RNA-binding site ligand fragment

39. Dockings at RNA-binding site RNA-binding site surface deeper ligand fragment

40. Ligand profiles at RNA-binding site • 2D structural similarity: ~35% for top 20 • Notable base fragment (present in 6 ligands) • Functional group: SO3 (present in 11 ligands) • H-bonding interactions: Ser72,Arg75, Tyr76, Lys79 • hydrophobic interactions/close contacts: Leu52, Phe87, Leu52, Asn82 • Molecular weight: 450-900 • Drug likeness: 20% (WDI)

41. Development of Databases for Screenings Maybridge 60,000 diverse Specs 10,000 diverse Pubchem 250,000 Chembridge 50,000 diverse NCI 1990 diverse Open collection 240,000 • All libraries converted into relational database format • Pubchem - 46,000 diverse library generated • Filtered based on lipinski’s rule of 5 • Redundancy checks performed Final database: 149,865 compounds with < 90% structural similarity

42. Example 4

43. Structural and functional dissection of the two nucleosome assembly proteins from Plasmodium falciparum Amit Sharma ICGEB, New Delhi

44. Importance Nucleosome assembly in P. falciparum

45. PfNAPS 1 42 217 269 N C 43 216 PfNAPL 1 46 298 359 N C 47 297 Workplan Nucleosome assembly in P. falciparum 1. Expressed in all stages of the parasite 2. Localized both to the cytoplasm and the nucleus 3. Differential localization in asexual/sexual stages 4. Differential phosphorylation of the two NAPz 5. Similar histone binding specificities

46. Nucleosome assembly in P. falciparum

47. Nucleosome assembly protein from P. falciparum

48. Nucleosome assembly protein from P. falciparum

49. Nucleosome assembly in P. falciparum

50. Example 5