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Structural dynamics of HER2 and ErbB4: Yin and Yang in
Mammary Carcinoma
Shannon TelescoAdvisor: 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-loopαC helix
N-loop
C-loop
A-loopαC helix
N-loop
C-loop
Active Inactive
Y877αC 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
Inactive HER2 Y877 Unphosphorylated
Active HER2 Y877 Unphosphorylated
Inactive HER2 Y877 Phosphorylated
Active HER2 Y877 Phosphorylated
F864 HN, E770 OE2
G865 HN, V842 O
G865 HN, H843 O
L866 O, R844 HE/HH11 L866 O, R844 HE/HH11
R868 HH12/22, D769 OD1/2
R868 HH12, R840 O
R868 HN/O, V842 O/HN R868 HN/O, V842 O/HN
L870 HN, R840 O L870 HN, R840 O
D871 O,
R840 HE/HH11/HH12
D873 OD1/2, R897 HE/HH22
E874 OE1/2, T759 HN/HG1
E876 OE1/2, R898 HH22/HE
Y877 O2/O3,
R844 HH/HH12/HH22 Y877 O2/O3,
R844 HH12/HH22
Y877 O3, K883 HZ1/2/3 Y877 O2, K883 HZ1/2/3
Y877 O2, R897 HH12/HH21
Y877 O3, R868 HH21/22
Y877 HN/O, F899 O/HN
A879 HN, R897 O
K883 HZ1/2/3, E757 OE1/2
V884 O, K887 HN
Salt Bridges
Hydrogen Bonds
Inactive HER2 Y877 Unphosphorylated
Active HER2 Y877 Unphosphorylated
Inactive HER2 Y877 Phosphorylated
Active HER2 Y877 Phosphorylated
D863, K753 D863, K753
E876, R898
D880, R897 D880, R897
K883, E766
Conserved bond
A-loop
Hydrogen bonds in the αC helix
Inactive HER2 Y877 Unphosphorylated
Active HER2 Y877 Unphosphorylated
Inactive HER2 Y877 Phosphorylated
Active HER2 Y877 Phosphorylated
E766, R756
E766, K883
E770, K753 E770, K753
Inactive HER2 Y877 Unphosphorylated
Active HER2 Y877 Unphosphorylated
Inactive HER2 Y877 Phosphorylated
Active HER2 Y877 Phosphorylated
A763 HN, S760 OG A763 HN, S760 OG
N764 HN, S760 O N764 HN, S760 O N764 HN, S760 O
E766 OE1/2,
R756 HH12/21/22/HE
D769 OD1/2, R868 HH12/22
E770 OE2, F864 HN
Y772 O, G776 HN Y772 O, G776 HN
V773 O, V777 HN V773 O, V777 HN
M774 O, L785 HN M774 O, L785 HN
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
R868E770K753
V842D863 (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)
RMSD (A-loop)
02468
1012141618
0 2 4 6 8 10 12 14
RMSD WRT Inactive (Å)
RM
SD
WR
T A
ctiv
e (Å
)
UnP-Active
P-Active
UnP-Inactive
P-Inactive
RMSD (αC helix)
0
2
4
6
8
10
12
0 2 4 6 8 10
RMSD WRT Inactive (Å)
RM
SD
WR
T A
ctiv
e (Å
)
UnP-Active
P-Active
UnP-Inactive
P-Inactive
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-loop C-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
R840V842
L870
R868Y877
R898
R897
A879E876
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
The dimerization interface
HER2: P707 Q711 M712 I714 L768 L790 V794
HER2 EGFRDimer interface
P707
V794
Y920
αC helix
N-terminal tail
EGFR: I917 Y920 M921 V924 M928 I929 V956
I917
Conformational shifts in heterodimer
RMSD for Dimer (αC helix)
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14
RMSD WRT Inactive (Å)
RM
SD
WR
T A
ctiv
e (Å
)
P-Y877 Dimer
UnP-Y877 Dimer
RMSD for Dimer (A-loop)
02468
1012141618
0 2 4 6 8 10 12 14 16
RMSD WRT Inactive (Å)
RM
SD
WR
T A
ctiv
e (Å
)
P-Y877 Dimer
UnP-Y877 Dimer
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
Inactive HER2 Y877 Unphosphorylated
Active HER2 Y877 Unphosphorylated
Inactive HER2 Y877 Phosphorylated
Active HER2 Y877 Phosphorylated
P707
Q711 Y772 Y772, G776
M712 Y772, L785 Y772, L785
I714
L768 N764, Y772 N764, D769 Y772 N764, E770
L790 N764
V794 N764 N764 T759 N764
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
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