Atomic resolution of the Molecular Mechanisms regulating the activation of the ErbB family Andrew Shih University of Pennsylvania Department 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 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.

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

Allosteric effects of Dimerization and Mutation

Symmetric Dimer Interface symmetric dimer interface residues stabilizing network residues proximal to symmetric dimer

Asymmetric Dimer Interface  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)

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 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.

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 RMSD to Active EGFR

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 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.

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

EGFR vs ErbB4 Primary Sequence  C-Helix EGFR 729 PRO LYS ALA ASN LYS GLU ILE LEU ASP GLU ALA TYR VAL MET ALA PRO LYS ALA ASN VAL GLU PHE MET ASP GLU ALA LEU ILE MET ALA 773 ErbB4 A-loop EGFR 831 ASP PHE GLY LEU ALA LYS LEU LEU GLY ALA GLU GLU LYS GLU TYR HSD ASP PHE GLY LEU ALA ARG LEU LEU GLU GLY ASP GLU LYS GLU TYR ASN 876 ErbB4 EGFR 847 ALA GLU GLY GLY LYS VAL ALA ASP GLY GLY LYS MET 882 ErbB4

EGFR vs ErbB4 Kinase Stabilizing Networks

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

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 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.

COREX/BEST Algorithm Hilser et al, Chem. Rev. (2006)

COREX/BEST Algorithm Hilser et al, Chem. Rev. (2006)

COREX/BEST Algorithm

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

Tables