Presentation on theme: "Structural dynamics of HER2 and ErbB4: Yin and Yang in Mammary Carcinoma Shannon Telesco Advisor: Ravi Radhakrishnan, Ph.D. Department of Bioengineering."— Presentation transcript:
Structural dynamics of HER2 and ErbB4: Yin and Yang in Mammary Carcinoma Shannon Telesco Advisor: Ravi Radhakrishnan, Ph.D. Department of Bioengineering
The HER2 signaling network Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB family of receptor tyrosine kinases (RTK). Ligand binding induces receptor dimerization and phosphorylation of tyrosine residues in the C-terminal tail segments. Tyrosines serve as docking sites for signaling molecules, activating molecular pathways such as cellular proliferation. Overexpression of HER2 results in ligand-independent activation and occurs in 20- 30% of human breast cancers. Yarden, Y and Sliwkowski, MX. Untangling the ErbB signalling network. Nat Rev Molecular Cell Biology 2001; 2:127-137.
Activation of ErbB tyrosine kinases Zhang, X., Gureasko, J., Shen, K., Cole, P., and Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 2006; 125:1137-1149. ErbB kinases are transmembrane receptors comprised of a ligand-binding extracellular domain, transmembrane segment, intracellular kinase domain, and tyrosine-rich C-terminal tail. Receptors can be auto- or transphosphorylated in their C-tails.
Structure of the HER2 kinase domain Activation loop (A-loop): Regulates accessibility of active site to binding Catalytic loop: Directly participates in phosphoryl transfer Nucleotide-binding loop (N-loop): Coordination of ATP & substrate tyrosine Alpha C helix: Facilitates coordination of substrate tyrosine
Regulation of HER2 activation Phosphorylation of Y877 in the A-loop may regulate extension of the loop and activation of HER2. A-loop αC helix N-loop C-loop A-loop αC helix N-loop C-loop ActiveInactive Y877 αC helix rotates into the active site
Elucidating HER2 activation mechanism Investigate the structural differences between inactive and active HER2. What are the key bonds that must be formed or broken upon activation? Define the role of Y877 phosphorylation in HER2 activation. Is HER2 unique from other ErbB members in that P-Y877 is necessary for activity? Predict the behavior of an EGFR/HER2 heterodimer. How might the dimerization interface trigger conformational changes in HER2?
Applying molecular dynamics (MD) to the HER2 system Four systems created: HER2 inactive & active, with & without Y877-phosphorylation. Systems were solvated & ionized (150 mM NaCl) & heated to 300 K. MD simulations performed for 10 ns. Trajectories analyzed for key hydrogen bonds and conformational changes. Solvated inactive HER2.
Hydrogen bonds in the A-loop Salt Bridges Hydrogen Bonds Conserved bond A-loop
Hydrogen bonds in the αC helix Salt Bridges Hydrogen Bonds Key salt bridge αC helix
Dual inhibition of the active state Inactive HER2: E770-K753 bond is inhibited Active HER2: Sequestering residues release E770 & K753 R868 E770 K753 V842 D863 (coordinating Asp) Key salt bridge
Stabilizing H-bonds in the active state E766 K883Active A-loop Inactive A-loop Active αC helix Inactive αC helix Key salt bridge in active HER2 is K883-E766 Connects the αC helix with the A-loop, stabilizing the helix in the active site Bond is conserved among ErbB family members: K851-E734 (EGFR), K856-E739 (ErbB4) Inactive/active HER2 (superimposed)
Analysis of conformational shifting All four systems are stable during 10 ns production run Slight movement of αC helix in active system No shifting of inactive toward active in short timescale RMSD for A-loop and αC helix (10 ns)
Effect of Y877 phosphorylation Many receptor tyrosine kinases, including the insulin receptor, require phosphorylation of their A-loops for full kinase activity. The EGFR family is unique in that A-loop phosphorylation appears to be unnecessary for activation. The role of A-loop phosphorylation in HER2 is controversial, as several studies have highlighted the importance of Y877 phosphorylation for kinase activity. Active A-loop Inactive A-loop Y877
Effect of Y877 phosphorylation N-terminal end of A-loopC-terminal end of A-loop Equilibrated P-active HER2 contains a network of hydrogen bonds which pin the A-loop to underlying regions of the kinase, maintaining the loop in its extended conformation. These fastening residues occur at both ends of the A-loop. R844 L866 R840 V842 L870 R868 Y877 R898 R897 A879 E876 F899
Role of Y877 in bridging the A-loop The phosphoryl group on Y877 bridges the fastening residues on either side of the A-loop. P-Y877 forms hydrogen bonds with residues at the N-terminal end of the A-loop, including K883, R844, and R868. Unphosphorylated active HER2 lacks this bridging mechanism. P-Y877 P-active HER2 (Activation loop) K883 R844 R868
Comparison between HER2 and insulin receptor tyrosine kinase Equilibrated P-active HER2 shares structural features with P-IRK. R1155 and P-Y1163 make VDW contacts in IRK. Likewise, R868 and P-Y877 form hydrogen bonds in HER2. Structure is unique to HER2 & IRK, as R868 is a lysine (K836) in EGFR. HER2 (P-active) Insulin RTK P-Y1163 (IRK) P-Y877 (HER2) R1155 (IRK) R868 (HER2) P-active HER2 superimposed on insulin RTK
Dimerization of HER2 ErbB kinases dimerize in an asymmetric head-to-tail configuration, similar to that seen for cyclin/cyclin- dependent kinase complexes. Monomer A is the activated kinase & monomer B is the activating kinase. The αC helix of monomer A comprises a key region of the dimerization interface. Zhang, X., Gureasko, J., Shen, K., Cole, P., and Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 2006; 125:1137-1149. Monomer B Monomer A
Constructing an EGFR/HER2 heterodimer Two different heterodimers were constructed: Y877-unphosphorylated inactive HER2 (activated kinase), active EGFR (activating kinase) Y877-phosphorylated inactive HER2 (activated kinase), active EGFR (activating kinase) Systems were solvated & ionized (150 mM NaCl) & heated to 300 K. MD simulations performed for 10 ns. Does dimerization promote activation of HER2? Is dimerization sufficient for activation or must HER2 also be phosphorylated? HER2 EGFR
Conformational shifts in heterodimer RMSD for dimeric HER2: αC helix and A-loop (10 ns) No significant movement of HER2 toward active form Shifting of αC helix due to adjustment to dimeric interface
Effect of dimerization on hydrogen bonding network Monomer A residues Residues in each of the four monomeric systems predicted to be affected by the dimerization interface: Bonds broken (Phosphorylated inactive HER2): M774-L785, E874-T759, G865-H843, R868-R840, and V884-K887 Bonds broken (Unphosphorylated inactive HER2): N764-S760, Y772-G776, G865-V842, D873-R897, and K883-E757
Conclusions Inactive and active HER2 structures reveal distinctive hydrogen bonding patterns that stabilize each conformation. A dual inhibitory mechanism maintains the inactive state through sequestration of key residues required for activation. Phosphorylation of Y877 may serve to bridge the stabilizing hydrogen bonds on either side of the A-loop in the active conformation. Unphosphorylated active HER2 lacks this bridging mechanism. Formation of EGFR/HER2 heterodimer results in repositioning of the αC helix and breakage of several key bonds that are present in the inactive state.
Part II: Role of ErbB4 signaling in the mammary gland
Opposing roles of HER2 and ErbB4 in breast cancer In contrast to HER2, expression of ErbB4 in breast cancer is associated with a favorable prognosis & a differentiating tumor phenotype. ErbB4 activation of STAT5a in the mammary gland regulates lactational expression of milk genes such as beta-casein. STAT5a is recruited to ErbB4 through binding of phosphotyrosine peptides by the SH2 domain. Williams, C., Allison, J.G., Vidal, G.A., Burow, M.E., Beckman, B.S., Marrero, L., and Jones, F.E. The ErbB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5a nuclear chaperone. JCB 2004; 167(3): 469-478.
PAINTing a picture of the interaction between ErbB4 and STAT5a Goal is to connect ErbB4 to its regulated transcription factors, such as STAT5a, by applying a bioinformatics program called PAINT. PAINT (Promoter Analysis & Interaction Network Toolset) is a computational tool which analyzes microarray data & generates networks connecting upregulated genes to their respective transcription factors. Given a list of genes (microarray data), PAINT can: Fetch potential promoter sequences for the genes in the list. Find Transcription Factor (TF) binding sites on the sequences. Analyze the TF-binding site occurrences for over/under-representation compared to a reference. Vadigepalli, R, Chakravarthula, P, Zak DE, Schwaber JS, and Gonye, GE. PAINT: a promoter analysis and interaction network generation tool for gene regulatory network identification. OMICS 2003; 7(3):235-53.
Bridging the genomics and atomistic scales in ErbB4 signaling Input microarray data from experiment Perform PAINT analysis to identify relevant TFs (Genomics Scale) Analyze key TF/binding partner interactions using molecular dynamics (Atomistic Scale) Validate structural predictions experimentally
Preliminary results: PAINT analysis The PAINT method was applied to the following study, which focused on stimulation of ErbB4 in mammary epithelial cells: Amin, DN, Perkins AS, and Stern DF. Gene expression profiling of ErbB receptor and ligand-dependent transcription. Oncogene 2004 Feb 19; 23(7):1428-38. In the study, agonistic antibodies as well as natural ligands (neuregulin) were used to activate the ErbB4 pathway, and ErbB4-stimulated gene expression was assessed by microarray analysis. Several novel ErbB4 gene targets were identified and their associated transcription factors were predicted by PAINT.
Preliminary results: PAINT analysis Genes (Color-Coded by Cluster) TF Red blocks correspond to over-representation of a TF in a given gene cluster. Cyan blocks correspond to under-representation of a TF in a given gene cluster. STAT5a
STAT5a interaction with ErbB4 Mao, X., et al. Structural bases of unphosphorylated STAT1 association and receptor binding. Mol Cell 2005; 17(6):761-71. STAT1 bound to phosphotyrosine peptide on IFN-γ receptor. The SH2 domain of STAT5a binds to P-Y959 at the C-terminal end of ErbB4’s kinase domain. Structural details of the SH2 domain-phosphotyrosine peptide interaction are known (STAT1-IFN-γ crystal structure). Can we predict features of the interaction between ErbB4 and STAT5a? STAT1 SH2 domain IFN-γ phosphopeptide
STAT5a interaction with DNA Chen, X., et al. Crystal structure of a tyrosine phosphorylated STAT1 dimer bound to DNA. Cell 1998; 93(5):827-39. Upon dimerization, STAT5a migrates to the nucleus and initiates transcription of genes containing GAS promoter sequences. Crystal structures of STAT1 and STAT3 bound to DNA reveal a nine base-pair consensus sequence. Monomers form a ‘pliers’-like structure in which dimerization is mediated by the SH2 domains. DNA SH2 domains
Predicting STAT5a interaction with ErbB4 and DNA To summarize, goals of ErbB4 study are two-fold: Predict binding of ErbB4 to STAT5a SH2 domain. Is it structurally possible for ErbB4 to phosphorylate the key tyrosine on STAT5a? Does ErbB4 bind to STAT5a as a monomer or as a dimer? Assess STAT5a binding to DNA consensus sequence. Which interactions regulate specificity of nucleotide-binding? What mutations in the DNA sequence or STAT5a DNA-binding domain abolish the interaction? Experimentally validate through mutagenesis studies and EMSA assays. Elucidate structural features of key interactions involved in the ErbB4 signaling pathway.
Acknowledgments Ravi Radhakrishnan, Ph.D. Mark Lemmon, Ph.D. Rajanikanth Vadigepalli, Ph.D. Andrew Shih
References Bose, R, Molina, H, Patterson, AS, Bitok, JK, Periaswamy, B, Bader, JS, Pandey, A, and Cole, PA. Phosphoproteomic analysis of Her2/neu signaling and inhibition. PNAS 2006; 103(26):9773-9778. Chen, X, Vinkemeier, U, Zhao, Y, Jeruzalmi, D, Darnell, JE Jr, and Kuriyan, J. Crystal structure of a tyrosine phosphorylated STAT1 dimer bound to DNA. Cell 1998; 93(5):827-39. Fiser, A, Sali, A. Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol 2003; 374:461-91. Hubbard, SR, Wei, L, Ellis, L, and Hendrickson, W.A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 1994; 372: 746-54. Lemmon, MA. The EGF receptor family as therapeutic targets in breast cancer. Breast Dis 2003; 18:33-43. Linggi, B, Cheng, QC, Rao AR, and Carpenter G. The ErbB4 s80 intracellular domain is a constitutively active tyrosine kinase. Oncogene 2006; 25: 160-63. Liu Y, Purvis J, Shih A, Weinstein J, Agrawal N, Radhakrishnan R. A multiscale computational approach to dissect early events in the Erb family receptor mediated activation, differential signaling, and relevance to oncogenic transformations. Ann Biomed Eng 2007; 35(6): 1012-25. Mao, X, Ren, Z, Parker, GN, Sondermann, H, Pastorello, MA, Wang, W, McMurray, JS, Demeler, B, Darnell, JE Jr, and Chen, X. Structural bases of unphosphorylated STAT1 association and receptor binding. Mol Cell 2005; 17(6):761-71. Schulze, WX, Deng, L, and Mann, M. Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Systems Biology 2005; 1:2005.0008. Stamos, J, Sliwkowski, MX, and Eigenbrot, C. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. JBC 2002; 277(48): 46265-46272.
References Stein, RA and Staros, JV. Insights into the evolution of the ErbB receptor family and their ligands from sequence analysis. BMC Evol Biol 2006; Oct 6; 6:79. Sundvall, M, Peri, L, Maatta, JA, Tvorogov, D, Paatero, I, Savisalo, M, Silvennoinen, O, Yarden, Y and Elenius K. Differential nuclear localization and kinase activity of alternative ErbB4 intracellular domains. Oncogene 2007; 26(48): 6905-14. Vadigepalli, R, Chakravarthula, P, Zak DE, Schwaber JS, and Gonye, GE. PAINT: a promoter analysis and interaction network generation tool for gene regulatory network identification. OMICS 2003; 7(3):235-53. Wang, SE, Narasanna, A, Perez-Torres, M, Xiang, B, Wu, FY, Yang, S, Carpenter, G, Gazdar AF, Muthuswamy, SK, and Arteaga, CL. HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell 2006; 10: 25-38. Williams, C., Allison, JG, Vidal, GA, Burow, ME, Beckman, BS, Marrero, L., and Jones, FE. The ErbB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5a nuclear chaperone. JCB 2004; 167(3): 469-478. Wood ER, Truesdale AT, McDonald OB, Yuan D, Hassell A, Dickerson SH, Ellis B, Pennisi C, Horne E, Lackey K, Alligood KJ, Rusnak DW, Gilmer TM, and Shewchuk L. A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res 2004; 64(18): 6652-9. Yarden, Y and Sliwkowski, MX. Untangling the ErbB signalling network. Nat Rev Molecular Cell Biology 2001; 2:127-137. Zhang, X., Gureasko, J., Shen, K., Cole, P., and Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 2006; 125:1137-1149.