Download HER2

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 2030% 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
• 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.
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.
Structure of the HER2 kinase domain
Alpha C helix:
Facilitates
coordination of
substrate tyrosine
Nucleotide-binding loop
(N-loop): Coordination
of ATP & substrate
tyrosine
Activation loop (A-loop):
Regulates accessibility of
active site to binding
Catalytic loop:
Directly participates
in phosphoryl transfer
Regulation of HER2 activation
αC helix rotates into the active site
Y877
Active
A-loop
αC helix
N-loop
C-loop
Phosphorylation of Y877 in the A-loop may regulate
extension of the loop and activation of HER2.
Inactive
A-loop
αC helix
N-loop
C-loop
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
Salt Bridges
Active HER2
Y877 Unphosphorylated
D863, K753
Inactive HER2
Y877 Phosphorylated
Active HER2
Y877 Phosphorylated
D863, K753
E876, R898
D880, R897
D880, R897
Inactive HER2
Y877 Unphosphorylated
Active HER2
Y877 Unphosphorylated
K883, E766
Inactive HER2
Y877 Phosphorylated
Active HER2
Y877 Phosphorylated
F864 HN, E770 OE2
G865 HN, V842 O
G865 HN, H843 O
Conserved bond
L866 O, R844 HE/HH11
L866 O, R844 HE/HH11
R868 HH12/22, D769 OD1/2
R868 HH12, R840 O
Hydrogen Bonds
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
A-loop
V884 O, K887 HN
Hydrogen bonds in the αC helix
Inactive HER2
Y877 Unphosphorylated
Active HER2
Y877 Unphosphorylated
Inactive HER2
Y877 Phosphorylated
Active HER2
Y877 Phosphorylated
E766, R756
Salt Bridges
E766, K883
E770, K753
Inactive HER2
Y877 Unphosphorylated
N764 HN, S760 O
Key salt bridge
Active HER2
Y877 Unphosphorylated
E770, K753
Inactive HER2
Y877 Phosphorylated
Active HER2
Y877 Phosphorylated
A763 HN, S760 OG
A763 HN, S760 OG
N764 HN, S760 O
N764 HN, S760 O
E766 OE1/2,
R756 HH12/21/22/HE
D769 OD1/2, R868 HH12/22
Hydrogen Bonds
E770 OE2, F864 HN
Y772 O, G776 HN
Y772 O, G776 HN
V773 O, V777 HN
M774 O, L785 HN
αC helix
V773 O, V777 HN
M774 O, L785 HN
Dual inhibition of the active state
R868
V842
K753
E770
D863 (coordinating Asp)
Key salt bridge
Inactive HER2:
E770-K753 bond is inhibited
Active HER2:
Sequestering residues
release E770 & K753
Stabilizing H-bonds in the active state
Active A-loop
Inactive A-loop
Active αC helix
Inactive αC helix
K883
E766
• 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
RMSD for A-loop and αC helix (10 ns)
RMSD (αC helix)
18
16
14
12
10
8
6
4
2
0
12
UnP-Active
P-Active
UnP-Inactive
P-Inactive
RMSD WRT Active (Å)
RMSD WRT Active (Å)
RMSD (A-loop)
10
UnP-Active
8
P-Active
6
UnP-Inactive
4
P-Inactive
2
0
0
2
4
6
8
10
RMSD WRT Inactive (Å)
12
14
0
2
4
6
8
10
RMSD WRT Inactive (Å)
• 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
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
Y877
Inactive A-loop
Effect of Y877 phosphorylation
• 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.
R868
L870
R840
Y877
L866
A879
V842
E876
R898
R844
N-terminal end of A-loop
R897
F899
C-terminal end of A-loop
Role of Y877 in bridging the A-loop
R868
• 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.
R844
K883
P-active HER2 (Activation loop)
• Unphosphorylated active HER2
lacks this bridging mechanism.
P-Y877
Comparison between HER2 and insulin
receptor tyrosine kinase
HER2 (P-active)
Insulin RTK
P-Y1163 (IRK)
P-Y877 (HER2)
R1155 (IRK)
R868 (HER2)
• 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.
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/cyclindependent 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.
Monomer B
Monomer A
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.
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
V794
EGFR: I917 Y920 M921 V924 M928 I929 V956
αC helix
Y920
P707
I917
N-terminal tail
HER2
Dimer interface
EGFR
Conformational shifts in heterodimer
RMSD for dimeric HER2: αC helix and A-loop (10 ns)
RMSD for Dimer (αC helix)
14
18
16
14
12
10
8
6
4
2
0
P-Y877 Dimer
UnP-Y877 Dimer
RMSD WRT Active (Å)
RMSD WRT Active (Å)
RMSD for Dimer (A-loop)
12
10
8
P-Y877 Dimer
6
UnP-Y877 Dimer
4
2
0
0
2
4
6
8
10
12
RMSD WRT Inactive (Å)
14
16
0
2
4
6
8
10
12
14
RMSD WRT Inactive (Å)
• 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
Residues in each of the four monomeric systems predicted to be
affected by the dimerization interface:
Inactive HER2
Y877 Unphosphorylated
Monomer A residues
P707
Q711
M712
I714
L768
L790
V794
Active HER2
Y877 Unphosphorylated
Y772
Y772, L785
N764, Y772
N764
N764
Inactive HER2
Y877 Phosphorylated
Active HER2
Y877 Phosphorylated
Y772, G776
Y772, L785
N764, D769
Y772
N764, E770
N764
T759
N764
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
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.
TF
Genes (Color-Coded by Cluster)
STAT5a
STAT5a interaction with ErbB4
IFN-γ phosphopeptide
• 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
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.
STAT5a interaction with DNA
• Upon dimerization, STAT5a
migrates to the nucleus and initiates
transcription of genes containing
GAS promoter sequences.
SH2 domains
• 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.
Chen, X., et al. Crystal structure of a
tyrosine phosphorylated STAT1 dimer
bound to DNA. Cell 1998; 93(5):827-39.
DNA
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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