INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY

(ICGEB), NEW DELHI, INDIA

Structural and Computational Biology Group

Organ Organ Tissue Tissue Cell Cell Molecule Molecule Atoms Atoms

Structural BiologyStructural BiologyMedicine and Biology at Atomic ScaleMedicine and Biology at Atomic Scale

High Resolution Structural BiologyHigh Resolution Structural Biology

Atomic structure - communicationAtomic structure - communication

Anti-tumor ActivityDuocarmycin SA

Evolution: Machine and ControlEvolution: Machine and Control

Atomic interactions

Biological linguisticsBiological linguistics

MoleculeStructural Genomics

PathwayStructural Proteomics

ActivitySystems Biology

RPARPA

NER

BER

RR

Techniques propel discoveriesTechniques propel discoveries

NMR Spectroscopy X-ray Crystallography

Computation

Atomic mapsAtomic maps

Experimental BuffetExperimental Buffet

RPA-B

RPA-AB

RPA-A

Flu

ores

cen

ce I

nte

nsi

ty

Ratio of T-ag/RPA

3D Molecular Structures3D Molecular Structures

X-ray

X-raysDiffraction

Pattern

Direct detection ofatom positions

Crystals

NMR

Indirect detection ofH-H distances

In solution

Flavours…Flavours…

X-ray- highest resolution, automation

NMR- enables solution variations;

direct tap on motions and on weak interactions

Computation- fundamentals of structure, dynamics

Structures breatheStructures breathe

Need to incorporate motion

Challenges…Challenges…

3D structures are static

• Biological process (recognition, interaction, chemistry) are dynamic

• New methods for molecular motions

Malaria

~40/400 are vectorsAnopheles gambiae

Plasmodium falciparumPlasmodium vivaxPlasmodium ovale

Plasmodium malaria

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 domainsfrom the erythrocytic stage

Invasion of erythrocytes by malaria parasites

Electron micrograph from Aikawa et al (1978) J. Cell Biol. 77:72

• apical orientation

• microneme secretion

• junction formation

• receptor/ligand interactions

• rhoptry discharge

RBC invasion

The Duffy-Binding-Like (DBL) Superfamily

SS TMI III - V VI CYTII (DBL)

F1 F2

P. vivax / P. knowlesi

P. falciparum

DBL1 DBL2 DBL3 DBL4CIDR1 Exon 2TM

Erythrocyte invasion: mediated by Erythrocyte binding protein family

Cytoadherence: mediated by PfEMP-1 family

P. falciparum

DBL

Erythrocyte

Erythrocyte

P. vivax RBC invasion P. falciparum RBC invasion

Duffy antigen

Sialic acid/GA

Sialic acid/GC

unknown

unknown

Overall domain architecture of PkDBL

DARC-PkDBL engagement

PolarSulfation of tyrosine 41 on DARC increases binding affinity ~1000X

Apolar

Polar

Invasion and evasion

Sialic acid binding siteCCR5 binding site

DARC binding site

Antigenic shift/drift by Conformational masking, Just-in-time release?sequence variation glycan shield, mutants

Haemagglutinin gp120 PkDBL

P. vivax versus P. falciparum

Structural and functional conservation - mechanistic divergence

F1 : F2 = 1.9Å (185 C) F1 : Pk-DBL = 1.6Å (195 C) F2 : Pk-DBL = 1.8Å (156 C)

Pk DBL Pf F1+F2 DBLs

Structural and functional conservation - mechanistic divergence

Insertions

Structural and functional conservation - mechanistic divergence

P. vivax/P. knowlesi P. falciparum

Module duplication

Insertions

Monomeric assembly Dimeric assembly

RBCRBC

RBC

Polymorphic sites

DARC binding site

Subdomain 3 loop

P. vivax Invasion P. falciparum

Example 2

Proteins that play crucial roles for the parasite

UIS3 from thepre-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

SH3 SH3PFG27

Novel foldUnique 3D structureRNA bindingSH3 bindingPxxP motifsSignaling intermediate

Gametocytogenesis in P. falciparum

Review of binding sites of interest on Pfg27

• Two RNA-binding sites per dimer

• Four SH3-binding sites per dimer

• A dimer interface

RNA-binding site Deep cavity

SH3-binding site Dimer interface

Pfg27 monomer

Docking pattern on Pfg27

Visual analysis of top 200 dockings

Deep cavity

RNA-binding site

Dimer interface

SH3-binding site

Other sites

FlexX GOLD

20 45

30 30

30 20

10 5

10 -

(values in percent)

RNA-binding site surface deeper

Docking at RNA-binding site

fragmentligand

Dockings at RNA-binding site

RNA-binding site surface deeper

fragmentligand

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

• All libraries converted into relational database format

• Pubchem - 46,000 diverse library generated

• Filtered based on lipinski’s rule of 5

• Redundancy checks performed

Specs

10,000 diverse

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 SharmaICGEB, New Delhi

Nucleosome assembly in P. falciparumImportance

47 297

1 46 298 359N C

1 42 217 269

43 216N C

PfNAPS

PfNAPL

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

Workplan

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

Funding agencies

Wellcome TrustEuropean Union

DBT, Govt. of IndiaICGEB

Manickam YogavelRachna Hora

Ashwani SharmaAnuj KumarJasmita Gill

Prakash MishraShoshanna Tharu

Tarun BhattManvi Gupta

Anupama YadavJ. Sebastian Raja