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
Title only.
You have just heard from Chris Wright and Randy Blakely about the their studies to understand how organs, tissues and cells work. Now I am going to take you deeper into the puzzle and zoom in on biology at the level of molecules and atoms. Along the way I will show you how the fundamental principles of math, physics, chemistry and computer science can be used to study complex biology. This information generates unique insights for understanding how to distinguish health and disease and is especially powerful for the development of new drug therapies.
So let’s zoom in.
FIRST ITEM
We all know that organs such as the brain or a kidney are collections of various brain and kidney tissues, which are themselves made up of brain and kidney cells. In continuing to zoom in with greater and greater and magnification on an organ, we eventually will see the millions of molecules that make up a cell. So how do all of these molecules work together to make up a functioning cell?
SECOND ITEM
Well, it turns out that communication is the key. If the molecules can all communicate properly, then the cell works just fine. But if something goes wrong in the communication, even with just one type of molecule, then the whole cell can be knocked out of kilter and set the stage for disease. Following this logic, it therefore makes sense to try and figure out how molecules communicate. Following the progression from organ to tissue to cell to molecule
THIRD ITEM
We need to zoom in on the molecules to study how they are made up. In fact, the key to understanding how molecules communicate is to determine the arrangements of their atoms. What is so fantastic about achieving this ultra high resolution view of biology is that it then becomes possible to understand how what distinguishes health and disease at its most basic level. This in turn means that we have a kind of magic bullet to target drugs to the specific molecules that are causing a disease, without inadvertantly hitting other molecules and causing side-effects.
LAST ITEM
It is this approach of studying the atomic structure of molecules important in biology that is termed Structural Biology, and last year I came here from the Scripps Research Institute in La Jolla, CA to create a new research program to bring this powerful technology to Vanderbilt and work with the outstanding researchers who make this such a successful institution.
Now, I would like to show you just one brief example of how Structural Biology works, in this case, how an extremely potent anticancer agent is targeted to a precise location on a molecule of DNA.

3. High Resolution Structural Biology
Atomic structure - communication

4. Evolution: Machine and Control
Anti-tumor Activity
Duocarmycin SA
Atomic interactions
To get a glimpse of the Structural Biology approach, I will now show you an example from the cancer research in our laboratory.
2nd SLIDE- TITLE
I will describe how taking a snapshot of the atomic structure of two molecules at the moment when they are communicating can be used to understand how an extremely potent anticancer agent is targeted to a precise location on a molecule of DNA.
 First item
In this picture the green and pink sticks represent the atomic structure of one small portion of a DNA sequence from a gene. If you look carefully, you can see the well-known features of the intertwined DNA double helix in these atoms.
2nd and 3rd item
The blue sticks that are placed within the cloud represent the atoms of the anticancer drug duocarmycin SA. The simple stick representation is used to help visualize the molecules but in fact, the cloud is a much more realistic representation of the atoms.
4th item
This picture show how well the drug and the DNA fit together, like a key in a lock. This gives a graphic representation of one of the key elements of designing a drug: the need to make the shape of the drug complementary the target. Many of the other key elements needed for drug design require an even deeper level of inspection of the structure of the molecule.
Next items- show small box and build the new larger box including the picture inside
We will zoom in closer to the atoms for a better view. At this level of magnification we can examine the details of the interactions between each atom in the drug and DNA target.
Next item- label “atomic interactions”
In this picture, the critical information is where the surface of the clouds come close to the sticks. It is here at this level of ultra magnification where we can really fine tune the details of the drug to generate the specificity that is needed to ensure there are no side effects to a drug.
Lights up to talk
Structural Biology is providing a whole new strategy for the design of drugs with higher specificity and fewer side effects than has been achievable with traditional approaches where hundreds or even thousands of random drug candidates must be scanned.
There is tremendous excitement in univeristies and the pharmaceutical industry because this structure-based drug design strategy will save huge amounts of time and money in the effort to develop new therapeutics for the clinic.
I look forward with anticipation to the opening of the new building when we will be placed in close juxtaposition with many of the most exciting laboratories on campus. I hope I have given you a sense of our excitement over the vast possibities of using the structural biology approach for advancing medicine and biology.

5. Biological linguistics
RPA
NER
BER RR
I dinked around w/colors, but there’s a plain white version at the very end if you prefer.
Also, see next slide for an animated version
Molecule
Structural Genomics
Pathway
Structural Proteomics
Activity
Systems Biology

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

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

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

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. Need to incorporate motion
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. Plasmodium falciparum
Malaria
Plasmodium falciparum
Plasmodium vivax
Plasmodium ovale
Plasmodium malaria
~40/400 are vectors
Anopheles gambiae

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

14. Example 1

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

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. Sialic acid binding site
Invasion and evasion
Haemagglutinin gp 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. knowlesi P. falciparum
Monomeric assembly Dimeric assembly
Module duplication
Insertions

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

27. Example 2

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

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. Gametocytogenesis in P. falciparum
SH3
PFG27
AUGCCUUA
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
From literature 3 sites have been identified by the group who have solved the crystal structure of pfg27. these sites are the RNA and SH3 binding sites. The dimer interface where the 2 monomers interact is also considered of potential interest, as the prevention of dimerisation of pfg27 could inhibit the activity of pfg27. from a visual inspection of the surface, the deepest cavity is also considered as a binding site.
Pfg27 monomer
RNA-binding site Deep cavity
SH3-binding site Dimer interface

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

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

39. Dockings at 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:
Drug likeness: 20% (WDI)

41. Development of Databases for Screenings
NCI 1990 diverse
Open collection 240,000
Pubchem
250,000
Chembridge
50,000 diverse
Maybridge
60,000 diverse
Specs
10,000 diverse
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. Nucleosome assembly in P. falciparum
Importance
Nucleosome assembly in P. falciparum

45. Nucleosome assembly in P. falciparum
Workplan
Nucleosome assembly in P. falciparum N C
PfNAPS
PfNAPL
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

51. Use Iodides for Phasing Protein Structures
using home Xray source

53. Example 6

59. Manickam Yogavel Rachna Hora Ashwani Sharma Anuj Kumar Jasmita Gill
Prakash Mishra
Shoshanna Tharu
Tarun Bhatt
Manvi Gupta
Anupama Yadav
J. Sebastian Raja
Funding agencies
Wellcome Trust
European Union
DBT, Govt. of India
ICGEB