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Natalia Jura
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Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3


Natalia Jura, Yibing Shan, Xiaoxian Cao, David Shaw, and John Kuriyan


December 9, 2009, doi: 10.1073/pnas.0912101106 (local copy)

Abstract / Figures from the paper / Supplemental Material / Coordinates



Abstract:

The kinase domain of human epidermal growth factor receptor (HER) 3/ErbB3, a member of the EGF receptor (EGFR) family, lacks several residues that are critical for catalysis. Because catalytic activity in EGFR family members is switched on by an allosteric interaction between kinase domains in an asymmetric kinase domain dimer, HER3 might be specialized to serve as an activator of other EGFR family members. We have determined the crystal structure of the HER3 kinase domain and show that it appears to be locked into an inactive conformation that resembles that of EGFR and HER4. Although the crystal structure shows that the HER3 kinase domain binds ATP, we confirm that it is catalytically inactive but can serve as an activator of the EGFR kinase domain. The HER3 kinase domain forms a dimer in the crystal, mediated by hydrophobic contacts between the N-terminal lobes of the kinase domains. This N-lobe dimer closely resembles a dimer formed by inactive HER4 kinase domains in crystal structures determined previously, and molecular dynamics simulations suggest that the HER3 and HER4 N-lobe dimers are stable. The kinase domains of HER3 and HER4 form similar chains in their respective crystal lattices, in which N-lobe dimers are linked together by reciprocal exchange of C-terminal tails. The conservation of this tiling pattern in HER3 and HER4, which is the closest evolutionary homolog of HER3, might represent a general mechanism by which this branch of the HER receptors restricts ligand-independent formation of active heterodimers with other members of the EGFR family.


Figures from the paper.

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First figure from paper

Figure 1: (A) Schematic diagram of EGFR domain structure and the ligand-dependent activation process. (B) Multiple sequence alignment of the portions of kinase domains of EGFR family members. The residues that are identical in all four sequences are highlighted in gray, with sequence alterations unique to HER3 highlighted in orange. Residues that are part of the receiver interface are marked by dark blue stars, residues at the activator interface are marked by pink dots, and residues participating in the binding of the JM-B segment are marked by green triangles.
Illustrator file, local only


First figure from paper

Figure 2: Crystal structure of the HER3 kinase domain. (A) Structure of the HER3 kinase domain in complex with AMP-PNP is compared with the crystal structure of the inactive EGFR kinase domain in complex with AMP-PNP (PDB ID code 2GS7) and the inactive HER4 kinase domain (PDB ID code 3BBW). (B) Detailed view of helix αC in the structure of HER3 (purple) and inactive EGFR (gray). (C) Detailed view of the ATP-binding site in the HER3 kinase domain, showing the electron density (at 3.5 σ above the mean value) around the AMP-PNP molecule and the metal ion. The difference electron density maps shown were calculated using a model of the protein at a stage before the inclusion of the nucleotide in the refinement.
Illustrator file, local only


First figure from paper

Figure 3: HER3 is catalytically impaired as a receiver kinase but can function as an activator kinase. Catalytic efficiency (kcat/KM) of the kinase constructs (residues 672–998 in EGFR and residues 674–1,001 in HER3) on vesicles is measured using the continuous coupled-kinase assay. The values of kcat/KM were obtained from the linear dependence of reaction velocity on substrate concentration at a low substrate concentration, and the error bars are derived from the linear fit (7).
Illustrator file, local only


First figure from paper

Figure 4: Analysis of the concatenated HER3 and HER4 kinase domains in the crystal lattices. (A) Pattern of kinase monomer interactions observed in the crystal lattices of the kinase domains of HER3 and inactive HER4 (PDB ID code 3BBW). The C-terminal tail exchanging dimers are propagated through the N-lobe dimer interface. JM refers to portion of the juxtamembrane segment. (B) Detailed view of the HER3 and HER4 N-lobe dimers. Hydrophobic residues are shown in stick representation. (C) Empirical estimates of binding free energy of HER3 and HER4 N-lobe homodimers using conformations obtained from all-atom molecular dynamics simulations. (D) Comparison between the C-terminal tail interaction with the C-lobe in the HER3 kinase domain dimer and the HER4 kinase domain dimer (PDB ID code 3BBW) and interaction of Mig6/segment 1 with the C-lobe of the EGFR kinase domain (PDB ID code 2RFE).
Illustrator file, local only


First figure from paper

Figure 5: Model for oligomerization of the kinase domains of HER3 and inactive HER4 at the plasma membrane. (A) Flexible C-terminal tail/C-lobe interaction creates a possibility for the formation of branched HER3 or inactive HER4 oligomers, resulting in a 2D mesh. (B) Empirical estimates of binding free energy of the HER3/HER4 N-lobe homodimer, calculated using conformations obtained from an all-atom molecular dynamics simulation.
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Supplemental Material

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First supplimental figure
Figure S1: HER3 is catalytically impaired towards the generic poly 4Glu:Tyr substrate. Catalytic efficiency (kcat/KM) of the kinase constructs (residues 672-998 in EGFR, and residues 674-1001 in HER3) on vesicles is measured using the continuous coupled kinase assay and the poly 4Glu:Tyr as a substrate. The values of kcat/KM were obtained from the linear dependence of reaction velocity on substrate concentration at low substrate concentration, and the error bars are derived from the linear fit.
Illustrator file, local only
First supplimental figure
Figure S2: The HER3 N-lobe dimer is structurally stable. (A) The interface of the HER3 N-lobe dimer is structurally stable in molecular dynamics simulations, as indicated by the moderate C- α atom RMSDs (with respect to the initial conformations) of the N-lobe β strands. (B) Accumulated water exposure at the interface in molecular dynamics simulations of the HER3 N-lobe homodimer (0.3 microseconds).
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First supplimental figure
Figure S3: In comparison to the N-lobe dimers, the non-specific HER3 dimeric contact is structurally less stable in molecular dynamics simulations. (A) Schematic and cartoon representation of the non-specific crystal contact in HER3 crystal lattice. (B) Accumulated water exposure at the interface in molecular dynamics simulations of the non-specific crystal HER3 crystal contact (0.12 microseconds). (C and D) Empirical estimate of C- α atom RMSDs (with respect to the initial conformations) of the N-lobe β strands (C) and binding free energy of HER3 non-specific crystal contact using conformations obtained from all-atom molecular dynamics simulations (D).
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First supplimental figure
Figure S4: Proposed oligomerization of the kinase domains of HER3 and inactive HER4 at the plasma membrane (A) The electrostatic surface potential calculated using APBS reveals regions of positive electrostatic potential on one face of the HER3 oligomer, suggesting an orientation of the oligomer at the plasma membrane. In this orientation the N-termini (extended by dashed lines) are located close to the plasma membrane, where they can connect to the transmembrane segments. (B) Empirical estimate of C- α atom RMSDs (with respect to the initial conformations) of the N-lobe β strands of HER3/HER4 non-specific crystal contact using conformations obtained from all-atom molecular dynamics simulations.
Illustrator file, local only


Coordinates


Coordinates in the Protein Data Bank:
2RFE
3BBW
2GS7