Journal of Biological Chemistry
Volume 288, Issue 35, 30 August 2013, Pages 25254-25264
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Signal Transduction
Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface*

https://doi.org/10.1074/jbc.M113.474882Get rights and content
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The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.

Background: HER2 and HER3 receptor tyrosine kinases form potent oncogenic signaling dimers.

Results: Carboxyl group footprinting and molecular dynamics reveal changes in the HER2-HER3 dimer interface and the HER2 activation loop.

Conclusion: HER2 and HER3 form asymmetric heterodimers in a single configuration. The HER2 unphosphorylated activation loop can assume an active conformation.

Significance: This study provides the first structural characterization of HER2-HER3 kinase dimers.

Epidermal Growth Factor Receptor (EGFR)
Mass Spectrometry (MS)
Molecular Dynamics
Protein-Protein Interactions
Receptor Tyrosine Kinase

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*

This work was supported, in whole or in part, by National Institutes of Health Grants R01CA161001 (to R. B.) and 8P41 GM103422-36. This work was also supported by National Science Foundation Grant NSF-DBI 0922879.

This article contains supplemental Table 1.

This article was selected as a Paper of the Week.

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Supported by National Institutes of Health T32 Training Grant 2T32HL007088-36.