Unveiling the Molecular Mechanisms Regulating the Activation of the ErbB Family Receptors at Atomic...

Unveiling the Molecular Mechanisms Regulating the Activation of the ErbB Family Receptors at Atomic Resolution through Molecular Modeling and Simulations

Andrew ShihUniversity of PennsylvaniaDepartment of Bioengineering

Advisor: Ravi Radhakrishnan

Receptor Tyrosine Kinase Structure and Function

Extracellular ligand binding domain: binds ligands from other cells to dimerize and active the RTK

Transmembrane domain and juxtamembrane domain: important in dimerization

Kinase domain: Transfers γ-phosphate of ATP to target molecules

Zhang et al, Cell (2006)

C-terminal Tail: Contains many phosphorytable residues and serves as docking for downstream signaling molecules

• The ErbB family is a set of four homologous receptor tyrosine kinases (RTK)

• RTKs are important in inter-cellular communication and consists of roughly four domains:

ErbB Family Network

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

Overall aim and Significance

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

Liu and Purvis, et al. Annals of Biomedical Engineering, 2007

The scope of this thesis is to understand the specific molecular mechanisms involved in the activation of ErbB1 (alternate name EGFR) and ErbB4 through modern computational biology techniques.

Specific Aims and Goals

The scope of this thesis is to understand the specific molecular mechanisms involved in the activation of ErbB1 (alternate name EGFR) and ErbB4 through modern computational biology techniques.

• 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. There is a network of stabilizing bonds that hold the inactive conformation stable and must be broken in order for a conformational change to occur.

• Aim 2: Characterize the effect of specific mutations (L834R and del S723-L728 ins S) and A-loop phosphorylation has upon the activation pathway in the EGFR kinase. Each stimuli causes a specific change in the activation pathway resulting in either a more active state or a more stable active state.

• Aim 3: Delineate the activation mechanism for the ErbB4 receptor tyrosine kinase, a kinase homologous to EGFRTK. The activation mechanism is qualitatively similar to EGFR, however there are minor differences in the specific residues and bonds to account for residue differences.

Specific Aims

• 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.

• Analyze bond patterns in monomer simulations to highlight gatekeeper residues and bonds in EGFR

• Examine the residues proximal to the dimer interfaces and clinically identified mutations in monomer simulations to identify those residues in the stabilizing network perturbed by the stimuli.

• Aim 2: Characterize the effect of specific mutations (L834R and del S723-L728 ins S) and A-loop phosphorylation has upon the activation pathway in the EGFR kinase.

• Aim 3: Delineate the activation mechanism for the ErbB4 receptor tyrosine kinase, a kinase homologous to EGFRTK.

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 phosphate binding loop (P-loop) and C-helix help position ATP and the target peptide

• Catalytic loop performs the phosphotransfer characteristic of kinases

Insulin Receptor Kinase

Hubbard et al, Nature, 1994

RTK Activation

Zhang et al, Cell (2006)

Canonically in RTKs, ligand binding causes a symmetric dimerization and auto-phosphorylation of the A-loop tyrosine causing activation.

EGFR was found to be unique in the A-loop tyrosine phosphorylation is not needed for activation. Gotoh et al, Biochem Biophys Res Comm, 1992

A proposed novel asymmetric dimer interface was found to initiate activation in EGFR. Specific mutations in the asymmetric dimer interface verified the interface by inhibiting kinase activity. Zhang et al, Cell, 2006

activated

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

• Following the simulation, the hydrogen bond patterns are analyzed and classified as stabilizing bonds (present in majority of the simulation).

Bond Hypothesis

The relative stability of different conformational states (and their associated relative free energies) is determined by a combination of several non-additive effects such as electrostatics, specific interactions, hydrophobic, solvation, and entropic contributions. Our hypothesis is based on the assumption that specific interactions dominate over the other effects in terms of discriminating between the active and inactive states.

So, to that end, we performed a hydrogen bond analysis upon the simulation trajectories and sorted out stabilizing bonds:

• Hydrogen bond: present in at least 60% of the trajectory and a bond length of 3.4 Å or less• Salt bridge: bond between a basic and an acidic residue with a bond length of 2.0 Å or less and present in 85-90% of the trajectory.

To help narrow the number of bonds looked at we focused the analysis on the four loops highlighted by Stamos et al, (P-loop, C-helix, C-loop and A-loop). The P-loop and C-loop analysis is not shown here since they are identical for most systems.

For now, we also focused external bonds of each individual loop (a bond between one residue within the specified loop and the other not within the specified loop).

Bond patterns in active and inactive unphosphorylated EGFR

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



The active and inactive conformations of EGFR have vastly different bond patterns.

To help find gatekeeper residues we paid close attention to those bonds and residues necessary for catalysis, highlighted in the crystal structure Stamos et al., J Biol Chem (2002)

• E738-K721: highly conserved salt bridge that helps coordinate ATP

• D831 (coordinating aspartate): aspartate also helps coordinate the ATP

• D813 (catalytic aspartate): residue which initiate phosphotransfer

Key Inactivating Network of bonds in EGFRTK

Comparing the EGFR bond networks, with key loops highlighted for the active Stamos et al, J Biol Chem (2002)

(yellow) and inactive (purple) Zhang et

al, Cell (2006).

Enhanced views of key gatekeeper bonds: K836 sequesters E738 and prevents the crucial E738-K721 salt bridge from forming (B and C).

L834 sequesters the catalytic aspartate (D813).

activeinactive

Landau et al, Structure, (2004)

Zhang et al, Cell, (2006)

Contrasting Allosteric Effects of Dimers in the Monomer System

By cross referencing the residues proximal to the dimer interfaces and those in the stabilizing bond network we can infer how each interface disrupts the stabilizing bonds

The symmetric dimer interface is not proximal to any stabilizing bond residues, showing little or no effect on the destabilization of the inactive state.

The asymmetric dimer interface has several residues proximal to stabilizing bond residues.

The residues are not directly bonded to any of the gatekeeper residues, but are mostly contained in the αC-helix.

Implies the activation mechanism forces the αC-helix to move

Symmetric Dimer Interface

Asymmetric Dimer Interface

Active InactiveW707E712K713K715I716Q767K822K828L977D979M983D984D985

Active InactiveV987D988A989D990E991Y992L993I994K799R938K946R949R953

sym

met

ric d

imer

in

terf

ace

resi

dues

stabilizing network residues proximal to symmetric dimer

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

N-lobe of the activated RTK

C-lobe of the activating RTK

asym

met

ric d

ime

r in

terf

ace

resi

due

s stabilizing network residues proximal to asymmetric dimer

sym

met

ric d

imer

in

terf

ace

resi

dues

Simulation of the asymmetric dimer interface

The following bonds break in the simulation Y740-S744, H846-R865, K851-R812 with the L834 HN-D13 OD1 H-bonds and K836-E738 salt bridge weakening significantly.

• Simulation of the asymmetric dimer

• One dimer is inactive (activated dimer) shown in blue and the activating dimer is left in the active state, as in the crystal structure.




asym

met

ric d

ime

r in

terf

ace

resi

due

s stabilizing network residues proximal to asymmetric dimer

activated kinase (inactive)

activating kinase (active)

dimer interface residues

Visualization of the asymmetric dimer interface effects

monomer inactive kinase dimer inactive kinase

comparison of A-loop and C-helix conformation of the dimer

A-loop

C-helix

Clinically Identified Mutants work in three different fashions

Affects dimerization (E685G and G695S)

Active InactveE685GG695Sdel L723-S728 insS S728

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

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

Affects key residues in stabilizing network (L837Q and L834R)

There are a set of clinically identified mutations, found in lung cancer patients that increase the basal kinase rates of EGFR.

By visualizing those residues both proximal to the mutations and those participating in the stabilizing network, we can classify the mutations into three categories

Created asymmetric dimer simulations of these mutants

Lynch et al, NEJM, (2004), Paez et al, Science (2004)

Conclusions and Completing the Aim

• The K836-E738 salt bridge and L834 HN-D813 OD1 H-bond are key sequestration bonds that need to be broken for activation

• The asymmetric dimer interface forces activation by αC-helix movement through allosteric contacts, breaking several of the stabilizing network bonds.

• The clinically identified mutations fall into three categories: affecting dimerization, affecting C-helix conformation and affecting key residues in the stabilizing bond network.

Completing the aim

• This aim is complete, however we are continuing the dimer simulation in hopes an activation event will be captured. Especially in the mutant dimer simulations.

Shih, A., Choi, S.H., Lemmon, M., and Radhakrishnan, R., To be Submitted, (2008).

Specific Aims



• Map the full activation pathway between inactive and active of the unphosphorylated system through umbrella sampling techniques.

• Use alchemical perturbation simulation techniques to quantify the energetic changes of the associated loop and residue conformation changes between the certain stimuli (L834R, del S723-L728 ins S and Y845 phosphorylation) and wildtype states.


Umbrella Sampling

Umbrella sampling allows the capturing of low probability events, in this case the full activation pathway.

Applies a series of harmonic potentials (umbrellas) to the system to create local minima and allow sampling there.

Afterwards use the weighted histogram algorithm methodology to remove the bias. Inactive

Active

EGFR Umbrella Sampling

We performed an initial 2-D umbrella sampling simulation for the fully solvated EGFR monomer kinase system with the following modified Hamiltonian

Where the reaction coordinate χ is the RMSD coordinate of the backbone atoms of the C-helix and A-loop with regards to the reference states. With ref. state 1 as the inactive crystal structure and ref. state 2 as the active crystal structure.

The reference points and are broken into specific windows to allow sampling along the entire activation pathway

RMSD to InactiveR

MS

D to

Act

ive I

A

EGFR Umbrella Sampling

Initial umbrella sampling simulation between the inactive and active conformations using the positions of the αC-helix (red) and the A-loop (blue) is the simultaneous reaction coordinates.

We know this initial reaction coordinate is a bad choice for umbrella sampling simulations.

Fully solvated system, solvent not shown

The activation pathway is straight path between the inactive and active state with a gap.

The gap implies the breakage of a key bond and then a cascade towards the active state.

The bond broken in the gap is the K836-E738 salt with the simultaneous formation of E738-K721

Examination at the end of the simulation shows the E848-R865 salt bridge is still holding the A-loop closed.

The key loop movements are held by key bonds

K836-E738

E738-K721

Inactive

E848-R865

Active

K836E738

K721

Inactive

K836

E738

K721

ActiveK836

E738K721

K836-E738 close to breaking

K836

E738K721

K836-E738 breaks E738-K721 forms

The αC-helix and A-loop move independently

• We have 35 ns of aggregate dimer simulations with similar results

• The tracking of the dimer systems shows the αC-helix is moving before the A-loop movement.

• Another simulation in lab has the reverse trend (active-> inactive) with the A-loop moving first.

• All this implies that the activation is a two-movement system, first the αC-helix moves towards active, then the A-loop moves towards active. With an intermediate structure between active and inactive where the αC-helix has moved, but the A-loop has not

E EE E

E

E


The initial umbrella sampling simulation was wrong.

New Hypothesis: First the EGFR activation pathway is a two step mechanism where the αC-helix and A-loop move independently, and the movements of these loops are reduced down to the specific salt bridges: αC-helix (breakage of K836-E738 and formation of E738-K721) and the A-loop (breakage of E848-R865).

Completing the aim

• Perform two separate umbrella sampling simulations for the movement of αC-helix and A-loop using the bond lengths specified as coordinates, to verify the new hypothesis.

• Perform the alchemical FEP for Y845 phosphorylation, L834R and del L723-S728 ins S

Alchemical Free Energy Perturbation

The alchemical free energy perturbation (FEP) allows the free energy change in mutations. It gradually changes the values of the simulation from the wildtype state to the mutant state.

Tyrosine to alanine test system (AYA->AAA). Unchanged atoms in green, initial atoms in red and final atoms in blue.

By combining the full energy pathway obtained before with the alchemical FEP we can get the energy change from the inactive to active mutants.

Specific Aims




• Identify the stabilizing network of bonds in each conformation to establish a mechanistic relationship between residues sequestering key activating residues and residues involved in the (symmetric and asymmetric) dimerization interface.

• Evaluate the possible dimer interfaces (i.e. symmetric, asymmetric) and the effect of the homologous ErbB1 clinically identified activating mutations in ErbB4 upon the stabilizing bonds

• Using alchemical free energy perturbation techniques, evaluate the free energy changes due to the L839R and R841A activating mutations

ErbB4 mutation predictions

In interest of time, a quick summary of the ErbB4 methods and results. The methodology for this aim is the same as Aim 1 (MD simulation, bond analysis, analyzing proximal residues to dimer interfaces and mutations).

The ErbB4 kinase is similar to the EGFR kinase in activation mechanisms. However, the ErbB4 system shows aDependence on a specific residue R841 (hom R836 in EGFR) not seen in EGFR. This is forced by a residue change from D853-E848 in ErbB4 to EGFR.

Mutation of this residue (R841A or R841E) should cause an activation of ErbB4, this mutation is not a homologously identified mutation from EGFR. A double mutant in EGFR, E848D and R841A/R841E should cause a similar activation.

ErbB4 EGFR

Completing the Thesis

High importance

•Complete the EGFR umbrella sampling using each of the key bonds as the reaction coordinate in a simulation.

• Analyze the proximal residues of the potential dimerization interfaces and verify the most likely interface using dimer simulations in ErbB4.

Low Importance

• Perform the alchemical FEP for Y845 phosphorylation, L834R and del L723-S728 ins S in EGFR.

• Perform alchemical FEP on ErbB4 on the L839R and R841A mutation to compare its affect to EGFR and validate results of bond network.

Thank you

Ravi Radhakrishnan

Committee Members

• Mark Lemmon

• Jeff Saven

• Casim Sarkar

Acknowledgements

• Yingting Liu

• Sung Hee Choi

Function of 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.

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

The ErbB4 kinase domain is cleaved following activation as a fully functional kinase dimer (s80). This allows direct correlation between the residues we highlight and those in cell biology/mutagenesis studies of the s80 fragment.

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 active Erbb4 active ErbB4 Inactive EGFR Inactive

E734 K851 E739 K856E739 R841

E738 K721 E743 K726E743 R841 E738 K836

N732 HN S728 O N737 HN G733 OD742 OD1, OD2 R841 HH11, 21

E738 OE1,2 F832 HN E743 OE2 F837 HNE743 OE1 R817 HH11, 12

Y740 O S744 HG1 L745 O S749 HN, HG1 L745 O S749 HN, HG1 Y740 O S744 HN,HG1 V741 O V745 HN I746 O M750 HN

M747 O L758 HN M742 O L753 HN A743 O L679 HN A748 O Q684 HN

A748 O R757 HE, HH12

D836 K726R841 E739R841 E743 K836 E738

E848 R812 D853 R870 D853 R870 E848 R865

K851 E734 K856 E739

D836 OD2 K726 HZ1, 3D836 OD1 T835 HG1

F832 HN E738 OE1,2 F837 HN E743 OE2G838 O R817 HE, HH12

L834 O R812 HH12 L834 HN D813 OD1

K836 HZ2 D737 OD2 R841 HH11, 21 D742 OD1, OD2 K836 O,HN V810 HN,O R841 HN, O V815 O, HN

L843 HN R813 O A840 HN G672 O

K848 O T873 HG1K848 HZ1, 2, 3 D937 OD1, 2E849 OE1, 2 K871 HZ1, 2, 3

Y845 O,HN Y867 HN,O Y850 HN, O F872 O, HN H846 NE2 R865 HH11

A852 HN R870 OK856 HZ1, 2, 3 E730 OE1, OE2

K851 O R812 HH11,21

alphaC-Helix (residues 729-743)

A-loop (Residues 831-852)

alphaC-helix (residues 729-743)

A-loop (residues 836-857) A-loop (residues 836-857)

alphaC-Helix (residues 734-748) alphaC-Helix (residues 734-748)

stabilizing H-bonds

stabilizing salt bridges

stabilizing H-bonds

stabilizing salt bridgesA-loop (residues 831-852)

• E743-K726: highly conserved salt bridge that helps coordinate ATP

• D836 (coordinating aspartate): aspartate also helps coordinate the ATP

• D818 (catalytic aspartate): residue which initiate phosphotransfer

We want to highlight

• D853-R870: salt bridge in EGFR hold A-loop closed

• R841: much more prevalent in ErbB4

Comparing EGFR vs ErbB4

ErbB4 bond network


• The ErbB4 system activates qualitatively the same is EGFR

• The D853-R870 salt bridge is maintained because of the longer side chain, forcing the prominence of R841

• Mutation of R841A would break the bonds and force activation. A double mutant of E848D and R836A would cause a similar activation

Completing the aim

• Analyze the proximal residues of the potential dimerization interfaces and verify the most likely interface using a dimer simulation.

• Perform alchemical FEP on the L839R and R841A mutation to compare its affect to EGFR and validate results of bond network.

Symmetric Dimerization Scheme


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

RTK Function and Activation


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)

Allosteric effects of Dimerization and Mutation

Allosteric effects of Y845 Phosphorylation

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

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

Asymmetric Dimer Interface


C-helix (residues 729 to 743)



asymmetric dimer interface residues

stabilizing network residues proximal to asymmetric dimer

ErbB4 Kinase domain is Cleaved


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.

The effects of Y845 phosphorylation needs more refined simulation techniques to characterize its effects.

Inactive EGFRTK

Effects of Y845 Phosphorylation

Src Stat5b

Regulates DNA synthesis

COXII

Effects internalization

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