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

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.

High Resolution Structural Biology Atomic structure - communication

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.

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

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

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

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

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

Need to incorporate motion Structures breathe Need to incorporate motion

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

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

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

Example 1

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

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

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

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

Overall domain architecture of PkDBLa

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

Sialic acid binding site 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

P. vivax versus P. falciparum

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)

Structural and functional conservation - mechanistic divergence Insertions

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

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

Example 2

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

Entry and Development – Liver Stages

Structural congruence, functional divergence

Genetics and structure driving insights into function

Genetics and structure driving insights into function

Genetics and structure driving insights into function

Example 3

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

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

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 20 45 30 30 30 20 10 5 10 - (values in percent)

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

Dockings at RNA-binding site surface deeper ligand fragment

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)

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

Example 4

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

Nucleosome assembly in P. falciparum Importance Nucleosome assembly in P. falciparum

Nucleosome assembly in P. falciparum Workplan Nucleosome assembly in P. falciparum 47 297 1 46 298 359 N C 1 42 217 269 43 216 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

Nucleosome assembly in P. falciparum

Nucleosome assembly protein from P. falciparum

Nucleosome assembly protein from P. falciparum

Nucleosome assembly in P. falciparum

Example 5

Use Iodides for Phasing Protein Structures using home Xray source

Example 6

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