atomic resolution of the molecular mechanisms regulating the activation of the erbb family andrew...

Atomic resolution of the Molecular Mechanisms regulating the activation of the ErbB family

Andrew ShihUniversity of PennsylvaniaDepartment of Bioengineering

Advisor: Ravi Radhakrishnan

Receptor Tyrosine Kinase (RTK) Structure and Function

Extracellular ligand binding domain

Transmembrane domain

Kinase domain

C-terminal Tail

Zhang et al, Cell (2006)

RTK Function and Activation

Citri and Yarden, Nat. Rev. Mol. Cell Bio. (2006)

ErbB Family Network

Yarden, Nat. Rev. Mol. Cell Bio. (2001)

Specific Aims and Goals

We aim to understand the activation pathways for ErbB1 (alternate name EGFR) and ErbB4 to better understand these crucial RTKs as well as RTKs in general

• Aim 1: Examine the molecular mechanisms governing the novel asymmetric kinase-kinase contact-mediated allosteric activation mechanism of the epidermal growth factor receptor tyrosine kinase.

• Aim 2: Characterize the effect of A-loop phosphorylation has upon the activation pathway in the EGFR kinase.

• Aim 3: Delineate the activation mechanism for the ErbB-4 receptor tyrosine kinase, a kinase homologous to EGFRTK.

• Aim 4: Multimeric protein-protein complexes of functional sub-units whose functionally is difficult to infer from the study of individual monomers.

Overarching goal is to link cell biology and crystallographic studies by analyzing molecular mechanisms of activation at the atomic level.

Specific Aims





Regulation of the Kinase domain

activation loop

C-helix

catalytic loop

nucleotide binding loop

N-terminal

C-terminal tail

• Activation loop (A-loop) is a short span of amino acids with at least one phosphorylatable residue (Y845 in EGFR) which regulates kinase activity

• Nucleotide binding loop (N-loop) and C-helix help position ATP and the target peptide

• Catalytic loop performs the phosphotransfer characteristic of kinases

Insulin Receptor Kinase

Dimerization Schemes

Zhang et al, Cell (2006)

Epidermal Growth Factor Receptor (EGFR) Activation

• Currently, EGFR does not activate in any conventional RTK fashion

• Does not dimerize in a symmetric fashion

• Mutation of A-loop tyrosine does not affect kinase activity

• Known activation stimulus for EGFR are

• Novel asymmetric dimer interface

• Several clinically identified activating mutations (E685G, G695S, del L723- S728 ins S, S744I, L837Q and L834R)

What mechanisms are governing the activation of EGFR and how are these stimulus affecting activation?

MD simulation

• Sum over all the potentials to get a potential for every atom in the system

• By differentiating the potentials for each atom we can obtain the force

• And advance each atom by one time step through

System Preparation

• Simulation of only the kinase domain

• Each system is explicitly solvated in 150 mM NaCl solution (Na+: yellow, Cl-: cyan, water: skyblue lines)

• The system is• minimized

• volume equilibrated• energy equilibrated• simulated for 10 ns

Inactivating Network of bonds in EGFRTK

Stabilizing network of bonds

GLU 734 LYS 851 GLU 738 LYS 721

GLU 738 LYS 836

ASN 732 HN SER 728 O GLU 738 OE1,2 PHE 832 HN

TYR 740 O SER 744 HG1 TYR 740 O SER 744 HN,HG1 VAL 741 O VAL 745 HN

MET 742 O LEU 753 HN ALA 743 O LEU 679 HN

LYS 836 GLU 738 LYS 843 ASP 932 GLU 848 ARG 812

GLU 848 ARG 865 LYS 851 GLU 734

PHE 832 HN GLU 738 OE1,2 LEU 834 O ARG 812 HH12

LEU 834 HN ASP 813 OD1 LYS 836 HZ2 ASP 737 OD2

LYS 836 O,HN VAL 810 HN,O LEU 838 HN ARG 808 O ALA 840 HN GLY 672 O

TYR 845 O,HN TYR 867 HN,O HSD 846 NE2 ARG 865 HH11

LYS 851 O ARG 812 HH11,21

alphaC-helix (Residues 729 to 743)Active Inactive

Stabilizing Salt Bridges

Stabilizing Stabilizing H-Bonds

Activation Loop (Residues 831 to 852)Active Inactive



Allosteric effects of Dimerization and Mutation

Symmetric Dimer Interface

Active InactiveW707E712K713K715I716Q767K822K828L977D979M983D984D985

Active InactiveV987D988A989D990E991Y992L993I994K799R938K946R949R953

symmetric dimer interface residues

symmetric dimer interface residues

stabilizing network residues proximal to symmetric dimer

stabilizing network residues proximal to symmetric dimer

Asymmetric Dimer Interface

Active InactiveP675L679 L679 A743 Y740 S744L680 L679 Y740 A743 Y740 L753I682L736 N732 D737 Y740 Y740L758V762 N732I917Y920M921V924M928I929V956

C-helix (residues 729 to 743)

Head RTK

Tail RTK

asymmetric dimer interface residues

stabilizing network residues proximal to asymmetric dimer

Clinically Identified Mutants work in three different fashions

•Affects dimerization (E685G and G695S)

•Affects C-helix conformation (del L723-S728 ins S and S744I)

•Affects stabilizing network (L837Q and L834R)

Active InactveE685GG695Sdel L723-S728 insS S728

S744I Y740 A743 S744 V745 Y740 S744L834R R812 L834 D813 L834L837Q K836 L838 K836

Completing the Aim

• Aim 1 is mostly complete, only requiring the analysis of dimer simulations to help validate the findings here done of single floating kinase studies.

Specific Aims





Effects of Y845 Phosphorylation

Src Stat5b

Regulates DNA synthesis

A-loop phosphorylation causes a large conformation shift in other RTKs (IRK, FGFR) and has importance in signaling pathways.

Aim2: How does Y845 Phosphorylation affect EGFR conformation at the atomic level?

Allosteric effects of Y845 Phosphorylation

Umbrella Sampling

WHAM Algorithm

Original Hamiltonian

Coupling Parameters

Biasing Potential

Histogram Function Reaction Coordinate

Samples per window

Free Energy Probability of being in a conformation without the biasing potential

• A statistical counting methodology that calculates free energy through probabilities• First changes the simulation data into a measure of the reaction coordinate and separates the data into histograms• Then calculates the probability the system will be in a conformation without the biasing potential• From these probabilities, WHAM calculates the free energies

Inverse Temperature

EGFR Activation Pathway

RMSD to Inactive EGFR

RM

SD

to

Act

ive

EG

FR

Completing the Aim

• A single umbrella sampling simulation has been done upon the Y845 unphosphorylated system. The areas need to be filled in, dependent upon analyses of each of the sampled section in the pathways (isolated WHAM analysis).

• The Y845 phosphorylated system will be simulated in a similar protocol and the results compared.

Specific Aims





Function of ErbB4

Aim 3: Delineate the activation mechanism for ErbB4 in a similar fashion to EGFR (Aim 1). Why ErbB4?

•ErbB4 is also a RTK, homologous to EGFR

• Unlike the rest of the ErbB family, ErbB4 is not over-expressed in cancers, but rather it is underexpressed.

• Recently studies have linked ErbB4 to the proper development of both the brain and the heart

• Furthermore ErbB4 is correlated with the onset of schizophrenia

• ErbB4 has two qualities useful to us. The amount of research on ErbB4 has been increasing in the last few years, which will help validate our studies. ErbB4 also has a novel property in the ErbB family.

ErbB4 Kinase domain is Cleaved


EGFR vs ErbB4 Primary Sequence

C-Helix EGFR729 PRO LYS ALA ASN LYS GLU ILE LEU ASP GLU ALA TYR VAL MET ALA 743759 PRO LYS ALA ASN VAL GLU PHE MET ASP GLU ALA LEU ILE MET ALA 773 ErbB4 A-loop EGFR831 ASP PHE GLY LEU ALA LYS LEU LEU GLY ALA GLU GLU LYS GLU TYR HSD 846861 ASP PHE GLY LEU ALA ARG LEU LEU GLU GLY ASP GLU LYS GLU TYR ASN 876 ErbB4 EGFR847 ALA GLU GLY GLY LYS VAL 852877 ALA ASP GLY GLY LYS MET 882 ErbB4

EGFR vs ErbB4 Kinase Stabilizing Networks

EGFR ErbB4

E734 K851 E764 K881 E738 K721 E768 K751

N732 HN S728 O N762 HN G758 O E738 OE1,2 F832 HN E768 OE2 F862 HN

Y740 O S744 HG1 L770 O S774 HN, HG1 V741 O V745 HN I771 O M775 HN A743 O L679 HN A773 O Q709 HN

D861 K751 K843 D932 E848 R812

D878 R895 K851 E734 K881 E764

D861 OD1 T860 HG1 F832 HN E738 OE1,2 F862 HN E768 OE2 L834 O R812 HH12

K836 HZ2 D737 OD2 K836 O,HN V810 HN,O R866 HN, O V840 O, HN L838 HN R808 O L868 HN R838 O A840 HN GLY 672 O

K873 O T898 HG1K873 HZ1, 2, 3 D962 OD1, 2E874 OE1, 2 K896 HZ1, 2, 3

Y845 O,HN Y867 HN,O Y875 HN, O F897 O, HNA877 HN R895 O

stabilizing salt bridges

stabilizing H-bonds

alphaC-Helix (residues 729-743) alphaC-Helix (residues 759-773)

A-loop (residues 831-852) A-loop (residues 861-882)

stabilizing salt bridges

stabilizing H-bonds

Symmetric Dimerization Scheme


Multimeric Protein Complexes

• The asymmetric dimer interface activates EGFR, however it is still unknown how the dimer forms following ligand binding.

• Similarly, Src is known to phosphorylate Y845 in EGFR but it is unclear when Src binds (before or after dimerization).

• Finally EGFR forms a ternary complex with Grb and SOS, we want to uncover whether there is a synergistic affect in their binding or not.

How does the binding of protein complexes affect the conformation of both proteins?

Completing the Aim

• The simulation of the unphosphorylated active ErbB4 is completed.

• The unphosphorylated inactive ErbB4 system has been constructed in homology with EGFR. However, the inactive conformation needs to be validated and possible alternative inactive conformations explored to reduce inaccuracies.

• The unphosphorylated inactive ErbB4 system then needs to be simulated and the bond tables compared in a similar fashion to Aim 1.

Specific Aims





COREX/BEST Algorithm

Hilser et al, Chem. Rev. (2006)

http://www.hbcg.utmb.edu/hilser/corexbest.htm


Hilser et al, Chem. Rev. (2006)

Completing the Aim

• The COREX/BEST algorithm provides us a methodology to examine long range protein fluctuations and has been validated by the Hilser group on small protein systems (SNAses).

• We need to map out the efficiency of the algorithm on a more complex biological system (EGFR) and analyze the effects of protein fluctuations in relation to a binding event.

Thank you

Acknowledgements

• Ravi Radhakrishnan

• Mark Lemmon

• Jeff Saven

Y845 Phosphorylation

In the active EGFRTK (not shown), there is no significant change in conformation or the stabilizing network.

The most salient change in the inactive EGFRTK is the extension of the C-helix, which in turn changes the stabilizing network to be more similar to the active EGFRTK.

Inactive EGFRTK

Y845 Phosphorylation

GLU 734 LYS 851 GLU 738 LYS 721

LYS 730 HZ1,2,3 GLU 848 OE1,2 ASN 732 HN SER 728 O ASN 732 HN SER 728 O

ASN 732 HD22 ALA 726 OASN 732 O VAL 762 HN

GLU 734 OE1,2 LYS 836 HZ1,3 GLU 738 OE1, OE2 LYS 836 HZ2

TYR 740 O SER 744 HN,HG1 TYR 740 O SER 744 HG1VAL 741 O VAL 745 HN VAL 741 O VAL 745 HN

ALA 743 O ARG 752 HH12

ASP 831 LYS 721 GLU 848 ARG 865

LYS 851 GLU 734

ASP 831 OD1 LYS 721 HZ1ASP 831 HN, O ASN 818 OD1, HD 21

PHE 832 HN GLU 738 OE2LEU 834 O ARG 812 HH12

LEU 834 HN ASP 813 OD1LYS 836 HZ1,3 GLU 734 OE1,2

LYS 836 HZ2 GLU 738 OE1, OE2 LYS 836 O,HN VAL 810 HN,OLEU 838 HN ARG 808 O TYR 845 O3 ARG 812 HH21TYR 845 O TYR 867 HN

ALA 847 HN ARG 865 O GLU 848 OE1,2 LYS 730 HZ1,2,3LYS 851 HZ1,2 GLU 734 OE1,2

alphaC-helix (Residues 729 to 743)Active Inactive


Stabilizing Stabilizing H-bonds



Activation Loop (Residues 831 to 852)Active Inactive

Tables

Active Inactive Active Inactive

2 0 2 1

6 6 5 2

Active Inactive Active Inactive

2 1 3 2

9 5 7 3

alphaC-helix

A-loop


Y845 Phosphorylated Y845 Unphosphorylated

Y845 Phosphorylated Y845 Unphosphorylated

Stabilizing H-bonds

Stabilizing H-bonds


PAS PIS UAS UISNumber of Atoms 109845 94945 57862 99184Charge of Protein -10 -10 -8 -8Number of Na 99 85 53 87Number of Cl 89 75 45 79

PAS UAS PIS UISE685GG695Sdel L723-S728 insS S728 S728 A726 S728 N732

S744I Y740 S744 V745 Y740 A743 S744 V745 A743 S744 V745 Y740 S744L834R R812 L834 R812 L834 D813 L834 D813 L834L837Q K836 L838 K836 L838 K836 K836

Residues Proximal to Clinical Mutations

PAS UAS PIS UISP675L679 L679 A743 Y740 A743 S744 Y740 S744L680 Y740 L679 Y740 A743 Y740 A743 Y740 L753I682L736 N732 Y740 V762 N732 D737 Y740 N732 Y740 Y740L758 V762V762 N732 V762 N732I917Y920M921V924M928I929V956

Residues Proximal to Dimer Interface

PAS UAS PIS UISLoopA-loop 0.5872 0.4746 0.7243 0.6604C-helix 0.3116 0.2723 0.3622 0.4015A-loop and C-helix 0.7865 0.6518 0.9299 0.9134

Entropy of specific loops (kcal/mol)

atomic resolution of the molecular mechanisms regulating the activation of the erbb family andrew...

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