Elodie Laine July 3 rd 2012, JOBIM, Rennes BiMoDyM, Molecular - - PowerPoint PPT Presentation

July 3rd 2012, JOBIM, Rennes

BiMoDyM, Molecular Oncology and Pharmacology Team, Labex LERMIT LBPA, CNRS-ENS de Cachan

Elodie Laine

518 protein kinases -2% of the proteome highly conserved through evolution highly oncogenic most studied pharmaceutical targets :

• ~ 10 approved inhibitors
• > 50 in clinical trials

PKA (1ATP)

Biological context

Receptor Tyrosine Kinases (RTKs) 20 subfamilies

2/22 Elodie Laine JOBIM– 03/07/2012

Receptor Tyrosine Kinases (RTKs)

KIT receptor

• Biological processes:

Proliferation, differenciation, survival…

• Pathologies :

Cancer, arthritis, Alzheimer’s disease

AMLs (10%), Mastocytoses (44%), GISTs (10%) GISTs (68%) Mastocytoses (>90%), Germinal tumors (26%), AMLs (9%)

Biological context

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Juxta-membrane region (JMR) Activation (A-) loop

In (1T45) Ac (1PKG)

Mutation D816V : mastocytoses + resistance to Imatinib

KIT inactive and active structures

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Role of KIT D816V mutation

A-loop JMR

WT MU D816V

JMR A-loop

50-ns molecular dynamics simulations local destabilisation (A-loop) + long-range re-organization (JMR)

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Role of KIT D816V mutation

facilitated JMR departure from PTK Release of an access to catalytic and substrate binding sites Leverage of JMR auto- inhibitory action is likely to trigger kinase activation X-ray structures

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Laine et al. 2011 PLOS Comput. Biol. Ac In

Short- and Long-range D816V effects

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?

How does the mutation of D816 into V induce a structural reorganization

• f a remote site distant by

more than 15 Å ?

WT MU D816V Hydrogen Bonds Hydrophobic Contacts

C-helix C-helix JM-S JM-S P/A-loops Prevalence (%)

100 80 60 40 20

• JMR-PTK interaction

network is altered

• A-loop and JMR

atomic fluctuations are modified

Interaction network & Atomic fluctuations

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Outline

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Biological Questions

• Information transmission

How do the mutation effects propagate between two distant sites of the protein ?

• From characterizing to designing

Can the dynamics of the protein, hence its function, be rationally modulated ?

Allosteric coupling Global conformational change

propagated through a pathway of well- defined interactions

Monod et al. 1965, J. Mol. Biol. Koshland et al. 1966, Biochem.

Local atomic fluctuations modification

propagated through multiple micro- dynamic pathways

Tsai et al. 2008, J. Mol. Biol. Schrank et al. 2009, PNAS

JOBIM– 03/07/2012

Outline

9/22 Elodie Laine

Biological Questions

• Information transmission

How do the mutation effects propagate between two distant sites of the protein ?

• From characterizing to designing

Can the dynamics of the protein, hence its function, be rationally modulated ?

Allosteric coupling Global conformational change

propagated through a pathway of well- defined interactions

Monod et al. 1965, J. Mol. Biol. Koshland et al. 1966, Biochem.

Local atomic fluctuations modification

propagated through multiple micro- dynamic pathways

Tsai et al. 2008, J. Mol. Biol. Schrank et al. 2009, PNAS hemoglobin purR

JOBIM– 03/07/2012

Principle of the method MONETA

Conformational Ensemble

All-atom molecular dynamics simulations M1 M2 M3 c2 c1 c6 c5 c4 c8 c3 c7

Modular Network Representation

residues clusters {M} linked by chains of residues {c}

hub

Dynamical Correlations + Topology

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What is a neighbor ?

• not adjacent in the sequence

range (i-1 ; i+1) forbidden

• connected by interaction(s)

Occupancy factor > 50%

• fast communication

CP < mean val [i-4 ; i+4]

Communication pathways

Commute times

CP(i,j) ~ < Δrij

TΔrij>

Chennubhotla & Bahar 2007

Communication pathways generation

• Start from a chosen residue xi
• Create as many paths as xi’s neighbors
• Grow path if the new neighbors

communicate fast with all the members of the current path Communication pathways represent chains of residues that mechanically transmit information

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Structural mapping of communication profile

The structural fragments of KIT that present communication hubs were previously reported as playing crucial functional roles in the activation/deactivation mechanisms of

• ther receptor tyrosine kinases or cytoplasmic kinases.

Per residue communication efficiency

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Modulation of KIT communication profile

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Communcation effciciency is enhanced ( ) or reduced ( ) upon D816V mutation

Independent dynamic segments

Principal Component Analysis (PCA)

Information by mode (global) Essential dynamics space (19-20 modes ~ 80 % fluct.) Information by atom (local) Most striking dynamic features (19-20 segments)

Local Feature Analysis (LFA)

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Prévalence (%)

100 80 60 40 20

Independent dynamic segments display the most striking features of the protein local dynamics

A-loop JMR Zhang & Wriggers, 2006

Independent dynamic segments

WT MU

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LFA correlation patterns

IDS-2 IDS-2 IDS-8 IDS-8 IDS-8 IDS-2 IDS-3 IDS-3 IDS-3

Relationship between IDS & CP

IDS are essentially isolated residues, hence IDS and CP are complementary

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Communication between A-loop and JMR

Communication routes are established in WT between the two regulatory segments, whereas in MU a decoupling is

• bserved

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WT MU

Design of a double mutant

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14ns 26ns 38ns 50ns dbMU

A-loop JMR

Atomic fluctuations A-loop

WT MU dbMU

ß-sheets 823-828 helix/turn 816-819

50-ns molecular dynamics simulations of D816V/D792E

Design of a double mutant

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Secondary structure assignments

Communication between A-loop and JMR

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MU dbMU WT

Communication between A-loop and JMR

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MU dbMU WT

The additional D792E mutation efficiently restored the communication as

• bserved in the wild type protein

Conclusion & Perspectives

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MOdular NETwork Analysis of protein structures

• Two-component modeling framework based on dynamical correlations and

topology of the protein that accounts for the duality of allosteric coupling

• Enables to localize and visualize information transmission throughout protein

structures and to rationally understand the determinants of signal propagation

• Applicability to KIT receptor:
• communication breakage put in evidence in D816V-mutated KIT
• design of a double mutant in which D816V long-range effect is neutralized
• in vitro measurements of wt, mu and dbmu mutant activation rates…?

 improvements of the method:

• other dynamical correlations measures
• more sophisticated algorithm for path generation

 application to drug design…?

• in vitro measurements of wt, mu and dbmu mutant activation rates…?

 improvements of the method:

• other dynamical correlations measures
• more sophisticated algorithm for path generation

 application to drug design…?

JOBIM– 03/07/2012

ByMoDyM, LBPA, ENS de Cachan, LabEx LERMIT (France) Luba Tchertanov Elodie Laine Manuela Leal da Silva Isaure Chauvot de Beauchêne Priscila da S. Figueiredo Celestino Florent Langenfeld Rohit Arora Samuel Demarest Loic Etheve Former members:

• S. Abdel Azeim
• J. André

Collaboration:

• E. Solary (IGR)
• P. Dubreuil (CRCM)
• F. Piazza (U. d’Orléans)
• A. Blondel (Institut Pasteur)
• B. Roux (U. of Chicago)
• P. BISCH (U. Federal do Rio de Janeiro)

Funding: OSEO-ISI ENS Cachan, France

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Acknowledgements

Poster n°65