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Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface*

  • Author Footnotes
    1 Supported by National Institutes of Health T32 Training Grant 2T32HL007088-36.
    Timothy S. Collier
    Footnotes
    1 Supported by National Institutes of Health T32 Training Grant 2T32HL007088-36.
    Affiliations
    From the Division of Oncology, Department of Medicine, and
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  • Karthikeyan Diraviyam
    Affiliations
    the Department of Biomedical Engineering and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109
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  • John Monsey
    Affiliations
    From the Division of Oncology, Department of Medicine, and
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  • Wei Shen
    Affiliations
    From the Division of Oncology, Department of Medicine, and
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  • David Sept
    Affiliations
    the Department of Biomedical Engineering and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109
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  • Ron Bose
    Correspondence
    To whom correspondence should be addressed: Div. Oncology, Dept. of Medicine, and Dept. of Cell Biology and Physiology, Washington University School of Medicine, Campus Box 8069, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-9308; Fax: 314-747-9308;
    Affiliations
    From the Division of Oncology, Department of Medicine, and

    the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and
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  • Author Footnotes
    * 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.
    1 Supported by National Institutes of Health T32 Training Grant 2T32HL007088-36.
Open AccessPublished:July 10, 2013DOI:https://doi.org/10.1074/jbc.M113.474882
      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.

      Introduction

      The epidermal growth factor receptor (EGFR)
      The abbreviations used are: EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; SASA, solvent-accessible surface area; MD, molecular dynamics; H-bond, hydrogen bond; A-loop, activation loop; GEE, glycine ethyl ester; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; AMP-PNP, adenylyl-imidodiphosphate; H/DX, hydrogen/deuterium exchange; Ni-NTA, nickel-nitrilotriacetic acid; PDB, Protein Data Bank.
      /ErbB family of receptor tyrosine kinases consists of four transmembrane receptor tyrosine kinases (EGFR/ErbB1, HER2/ErbB2/neu, HER3/ErbB3, and HER4/ErbB4) that play important roles in breast, lung, and other cancer types. Kinase activity is initiated by the binding of growth factor ligands (i.e. epidermal growth factor, neuregulin, etc.) to the extracellular domain of one or more EGFR family monomers and their subsequent homo- or heterodimerization. Dimerization of the extracellular domain brings the cytoplasmic kinase domains together, allowing the formation of asymmetric kinase domain dimers required for kinase activation (
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      An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.
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      Architecture and membrane interactions of the EGF receptor.
      ). The formation of different dimer pairs initiated by specific ligands allows a small number of proteins to affect several potential signaling pathways. The HER2-HER3 dimer pair represents a uniquely potent combination within this system. The HER2 receptor does not bind ligand. HER3, due to numerous substitutions in its catalytic domain, has significantly lower kinase activity and can only signal as a member of a heterodimer (
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      • Yarden Y.
      The deaf and the dumb: the biology of ErbB-2 and ErbB-3.
      ). Despite their individual limitations, the HER2-HER3 dimer forms the most potent receptor pair of the ErbB family in terms of cellular proliferation and transformation (
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      • Barbas 3rd, C.F.
      • Hynes N.E.
      The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation.
      ).
      HER2 is overexpressed in 20–30% of breast cancers (
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      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      ) and is the preferred heterodimerization partner of all other ErbB family members, especially HER3 (
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      A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.
      ,
      • Graus-Porta D.
      • Beerli R.R.
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      ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.
      ,
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      • Chen X.
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      • Ratzkin B.J.
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      ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.
      ). Therefore, understanding the inter- and intramolecular interactions of HER2 heterodimers is a high priority. In the case of most high resolution structures of ErbB family kinase domains, the structures are of homodimers (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
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      An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.
      ,
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      ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation.
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      Mechanism of activation and inhibition of the HER4/ErbB4 kinase.
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      • Jura N.
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      Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3.
      ). To date, the structure of an ErbB family kinase heterodimer has yet to be solved. Although crystallography has provided atomic level resolution of these kinases, it is limited in its potential to characterize dynamic structural events in a membrane environment.
      Several mass spectrometry (MS)-based footprinting strategies have been developed to provide insights into protein structure. These approaches include hydrogen/deuterium exchange (H/DX) (
      • Wales T.E.
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      Hydrogen exchange mass spectrometry for the analysis of protein dynamics.
      ) and hydroxyl radical labeling (
      • Xu G.
      • Chance M.R.
      Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.
      ). H/DX labels amide hydrogens, whereas hydroxyl radical labeling modifies aromatic, aliphatic, and sulfur-containing side chains. MS footprinting strategies require less protein material than traditional structural methods and have been successfully employed in the characterization of protein-protein interactions, oligomerization, and folding dynamics (
      • Konermann L.
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      • Pan Y.
      Protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches.
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      SUPREX (stability of unpurified proteins from rates of H/D exchange) analysis of the thermodynamics of synergistic anion binding by ferric-binding protein (FbpA), a bacterial transferrin.
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      Targeting Bcr-Abl by combining allosteric with ATP-binding site inhibitors.
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      • Rempel D.L.
      • Grayson M.A.
      • Remsen E.E.
      • Gross M.L.
      Application of SIMSTEX to oligomerization of insulin analogs and mutants.
      ). These techniques suffer some limitations when investigating membrane-associated proteins, such as extensive back-exchange (in the case of H/DX) or nonspecific oxidation (in the case of hydroxyl radical labeling) resulting from the extensive post-labeling sample cleanup required to remove lipids. However, recent improvements show promise for the implementation of these strategies for membrane proteins (
      • Hebling C.M.
      • Morgan C.R.
      • Stafford D.W.
      • Jorgenson J.W.
      • Rand K.D.
      • Engen J.R.
      Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.
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      • Stafford D.W.
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      Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs.
      ).
      An alternative to HD/X and hydroxyl radical footprinting is labeling carboxylic acid side chains (Glu and Asp) with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated incorporation of glycine ethyl ester (GEE) (
      • Hoare D.G.
      • Olson A.
      • Koshland D.E.
      The reaction of hydroxamic acids with water-soluble carbodiimides. A Lossen rearrangement.
      ,
      • Hoare D.G.
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      A method for the quantitative modification and estimation of carboxylic acid groups in proteins.
      ). This carboxyl group footprinting reaction is efficient in biologically relevant buffer systems and can tolerate the extensive post-labeling purification required for studying membrane protein structural dynamics. It can also be utilized to rapidly characterize several experimental conditions in parallel. We previously utilized this strategy to examine the dimer interface of the HER4/ErbB4 homodimer, determine its dimer association constant on a lipid membrane, and characterize the conformational changes resulting from activation loop tyrosine phosphorylation (
      • Zhang H.
      • Shen W.
      • Rempel D.
      • Monsey J.
      • Vidavsky I.
      • Gross M.L.
      • Bose R.
      Carboxyl-group footprinting maps the dimerization interface and phosphorylation-induced conformational changes of a membrane-associated tyrosine kinase.
      ).
      In this work, we utilize carboxyl group footprinting MS to examine the effect of dimerization and phosphorylation on recombinantly expressed HER2 and HER3 kinase domains. In vitro, the kinase domain interactions are weak and do not occur readily in solution, but they occur readily when the kinases are anchored to a surface. Using a previously described in vitro model system, dimerization was achieved using an N-terminal His6 tag to orient the kinase domain monomers on the surface of a nickel-chelating liposome, increasing the local concentration and facilitating protein-protein interaction (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.
      ,
      • Monsey J.
      • Shen W.
      • Schlesinger P.
      • Bose R.
      Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.
      ). Carboxyl group footprinting confirms the orientation of the heterodimer with HER2 and HER3 occupying the acceptor and donor positions, respectively. Additionally, we observed conformational changes in the HER2 activation loop upon heterodimerization and the addition of either ATP or its nonhydrolyzable analog AMP-PNP. We observed similar activation loop conformational changes in the nonreceptor tyrosine kinase Src using carboxyl group footprinting MS. We performed molecular dynamics simulations on a model of the HER2-HER3 asymmetric dimer and identified several inter- and intramolecular interactions. Finally, we performed functional analyses on footprinted residues and cancer-associated mutations, evaluating them within the context of the new structural model. These results provide the first structural insights into an ErbB family heterodimer and advance the understanding of ErbB family kinase activation.

      DISCUSSION

      This study provides the first structural characterization of HER2-HER3 asymmetric heterodimers on a lipid membrane surface. Carboxyl group footprinting MS confirms the asymmetric dimer model in which HER3 and HER2 are configured as the donor and acceptor kinases, respectively. A-loop motion, associated with HER2 activation, is not exclusively governed by tyrosine phosphorylation, but it occurs as a result of dimerization and nucleotide binding. Determining protein structure and motion is an important aspect of characterizing protein complexes and informing the design of inhibitors. Molecular dynamics simulations of the HER2-HER3 dimer model support carboxyl group footprinting MS results and identify key salt bridges at the heterodimer interface. MD has implicated several intra- and intermolecular interactions that provide additional stability to the open, nonphosphorylated A-loop conformation. Combining existing crystal structures with the solution-based information obtained with carboxyl group footprinting MS and the atomistic information provided by MD simulations produces a powerful platform for characterizing protein structural dynamics.
      HER2 has been shown to be the preferred heterodimerization partner for other ErbB family members, especially HER3 (
      • Tzahar E.
      • Waterman H.
      A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.
      ,
      • Graus-Porta D.
      • Beerli R.R.
      • Daly J.M.
      • Hynes N.E.
      ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.
      ,
      • Karunagaran D.
      • Tzahar E.
      • Beerli R.R.
      • Chen X.
      • Graus-Porta D.
      • Ratzkin B.J.
      • Seger R.
      • Hynes N.E.
      • Yarden Y.
      ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.
      ). Our analysis of an equimolar mixture of HER2 and HER3 also suggests that heterodimer formation is preferred over the formation of HER2-HER2 or HER3-HER3 homodimers. The carboxyl group footprinting experiments performed on HER2-HER3 dimers confirm the hypothesis that HER3 functions as the donor kinase and HER2 as the acceptor in an asymmetric configuration. Decreased GEE labeling was observed on acidic residues located near the dimer interface of HER3's C-lobe and HER2's N-lobe, including decreased GEE labeling of HER2 Glu-717 and HER3 Glu-909, which in MD simulations are involved in salt bridge interaction with HER3 Lys-907 and HER2 Lys-716, respectively (Fig. 6). Analogous salt bridge interactions were previously observed in HER4 homodimers (
      • Zhang H.
      • Shen W.
      • Rempel D.
      • Monsey J.
      • Vidavsky I.
      • Gross M.L.
      • Bose R.
      Carboxyl-group footprinting maps the dimerization interface and phosphorylation-induced conformational changes of a membrane-associated tyrosine kinase.
      ). Hydrophobic interactions also play a key role at the dimerization interface, and we will further explore those interactions in the future (
      • Telesco S.E.
      • Radhakrishnan R.
      Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study.
      ).
      The A-loop regulates access of target substrates to the catalytic loop, which facilitates phosphoryl transfer. Upon kinase activation, the A-loop undergoes a dramatic conformational shift in which it extends to uncover the kinase substrate-binding pocket (Fig. 3D) (
      • Huse M.
      • Kuriyan J.
      The conformational plasticity of protein kinases.
      ,
      • Stamos J.
      • Sliwkowski M.X.
      • Eigenbrot C.
      Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor.
      ). In many protein kinases, including the Src family of tyrosine kinases, the A-loop extension is stabilized by phosphorylation of a tyrosine residue located within the loop itself and is required for kinase activation (
      • Porter M.
      Reciprocal regulation of Hck activity by phosphorylation of Tyr527 and Tyr416. Effect of introducing a high affinity intramolecular SH2 ligand.
      ). The study of the Src family kinase Lck crystal structure, in which Tyr-394 on the activation loop was phosphorylated, shows the A-loop in an open and extended conformation with a hydrogen bonding network extending from the DFG aspartate to the phosphotyrosine, including interactions with the catalytic loop and αC-helix (
      • Yamaguchi H.
      • Hendrickson W.A.
      Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation.
      ). However, the requirement for A-loop tyrosine phosphorylation in EGFR and HER2 is still controversial (
      • Gotoh N.
      • Tojo A.
      • Hino M.
      • Yazaki Y.
      • Shibuya M.
      A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor.
      ,
      • Biscardi J.S.
      c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function.
      ,
      • Zhang H.T.
      • O'Rourke D.M.
      • Zhao H.
      • Murali R.
      • Mikami Y.
      • Davis J.G.
      • Greene M.I.
      • Qian X.
      Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential.
      ,
      • Segatto O.
      • Lonardo F.
      • Pierce J.H.
      • Bottaro D.P.
      • Di Fiore P.P.
      The role of autophosphorylation in modulation of erbB-2 transforming function.
      ,
      • Xu W.
      • Yuan X.
      • Beebe K.
      • Xiang Z.
      • Neckers L.
      Loss of Hsp90 association up-regulates Src-dependent ErbB2 activity.
      ,
      • Bose R.
      • Molina H.
      • Patterson A.S.
      • Bitok J.K.
      • Periaswamy B.
      • Bader J.S.
      • Pandey A.
      • Cole P.A.
      Phosphoproteomic analysis of Her2/neu signaling and inhibition.
      ). Previous molecular dynamics studies of homology-modeled HER2, based on the EGFR crystal structure, similarly suggested that a hydrogen bond network secured the open A-loop conformation upon tyrosine phosphorylation (
      • Telesco S.E.
      • Radhakrishnan R.
      Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study.
      ). The presence of several aspartic and glutamic acid residues along the activation loop makes carboxyl group footprinting MS a powerful technique for monitoring its conformational changes. Carboxyl group footprinting analysis of Src revealed a marked increase in the GEE labeling of A-loop acidic residues in the presence of ATP, indicative of the open A-loop observed in crystallographic studies. We observe an almost identical increase in HER2 A-loop modification when dimerized with HER3 in the presence of ATP, suggesting a similar open and extended A-loop conformation as that observed in Src. In contrast to Src, however, we also observe increased A-loop labeling with the addition of AMP-PNP, a nonhydrolyzable ATP analog. We thus conclude that the open A-loop conformation in HER2 is dependent on dimerization and nucleotide binding, but it does not require A-loop tyrosine phosphorylation.
      Molecular dynamics simulations were able to explore interactions that play a role in stabilizing the open conformation of the unphosphorylated HER2 activation loop. Several H-bond interactions are implicated in the open conformation. Interactions between the HER2 αC-helix and the A-loop, including Glu-766–Arg-868, and interactions involving Lys-753 and Asp-863 with the coordination with Mg2+ and ATP in the catalytic pocket have been previously described and also appear in our simulations (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.
      ,
      • Parang K.
      • Till J.H.
      • Ablooglu A.J.
      • Kohanski R.A.
      • Hubbard S.R.
      • Cole P.A.
      Mechanism-based design of a protein kinase inhibitor.
      ). We also observe H-bond interactions involving Asp-845, Arg-849, and Asn-850 in the catalytic loop that have been implicated in coordination and transfer of the γ-phosphate of ATP (Fig. 6C) (
      • Telesco S.E.
      • Radhakrishnan R.
      Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study.
      ,
      • Parang K.
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      • Kohanski R.A.
      • Hubbard S.R.
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      Mechanism-based design of a protein kinase inhibitor.
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      Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis.
      ). MD simulations also revealed novel interactions that stabilize the unphosphorylated A-loop of HER2. A persistent backbone-backbone interaction between Tyr-877 and Phe-899 is likely to be further stabilized by the parallel-displaced π-stacking of the two aromatic side chains, and interaction is not accounted for by the simulation that would provide 0.5–1.0 kcal/mol more stability (Fig. 6E) (
      • McGaughey G.B.
      π-Stacking interactions. Alive and well in proteins.
      ). We found interactions between several groups of residues, Asp-769, Tyr-772, and Asp-871 on HER2 with Asp-920, Arg-948, and Arg-951 on HER3, provide a possible mechanism for intermolecular stabilization of the open and extended activation loop (see, for example, Fig. 6D). Near the C-terminal end of the activation loop, several H-bonds may form between the extended A-loop and the C-lobe of the kinase domain. The implication of these interactions on the regulation of kinase activity warrants further study.
      The development of this new HER2-HER3 dimer structural model provides a new framework for interpreting mutation data. The HER2 kinase domain mutations D769H and D769Y have been observed in breast, lung, gastric, and colorectal cancers (
      • Bose R.
      • Kavuri S.M.
      • Searleman A.C.
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      • Shen D.
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      • Goel N.
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      • Ding L.
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      • Ellis M.J.
      Activating HER2 mutations in HER2 gene amplification negative breast cancer.
      ,
      • Lee J.W.
      • Soung Y.H.
      • Kim S.Y.
      • Nam S.W.
      • Park W.S.
      • Wang Y.P.
      • Jo K.H.
      • Moon S.W.
      • Song S.Y.
      • Lee J.Y.
      • Yoo N.J.
      • Lee S.H.
      ERBB2 kinase domain mutation in the lung squamous cell carcinoma.
      ,
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      • Soung Y.H.
      • Seo S.H.
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      • Park W.S.
      • Kim S.H.
      • Lee J.Y.
      • Yoo N.J.
      • Lee S.H.
      Somatic mutations of ERBB2 kinase domain in gastric, colorectal, and breast carcinomas.
      ). We recently reported that D769H/D769Y were activating mutations in breast cancer and possessed greater specific activity when assayed as HER2 homodimers (
      • Bose R.
      • Kavuri S.M.
      • Searleman A.C.
      • Shen W.
      • Shen D.
      • Koboldt D.C.
      • Monsey J.
      • Goel N.
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      • Li S.
      • Ma C.X.
      • Ding L.
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      • Ellis M.J.
      Activating HER2 mutations in HER2 gene amplification negative breast cancer.
      ). The kinase assay data performed in this work show that both D769H and D769Y are more activating relative to wild type in the context of the HER2-HER3 heterodimer (Fig. 7A). Furthermore, D769A results in a loss of kinase activity. Potential interpretations of these results include increased hydrophobic interaction between HER2 D769H/D769Y and HER3 Ile-919 and Met-923 or intramolecular interactions with the HER2 A-loop. Our MD simulations show the interaction of Asp-769 with the amide proton of HER2 Leu-869 in the A-loop, providing additional stability to the open conformation. Mutation to histidine or tyrosine increases the side chain length and hydrophobicity, which could provide additional stability to the open activation loop through interactions with the HER2 Leu-869 side chain. This additional stability accounts for the observed increases in D769H/D769Y monomer and dimer activity relative to wild type (Fig. 7A). In contrast, replacing the aspartate with a shorter nonpolar alanine would result in a loss of those stabilizing interactions. The corresponding loss of kinase activity for D769A supports this reasoning.
      Our molecular dynamics simulations suggest the interaction of HER3 Arg-948 with HER2 Asp-871 plays a role in the stabilization of the open and extended HER2 A-loop (Fig. 6D). We assessed the effect of this interaction on dimer activity by generating a HER3 R948A mutant. Compared with wild type HER3, R948A showed a modest but statistically significant decrease in kinase activity (Fig. 7C). We also assessed the effect of a R948K mutant, which has been observed in several cancer cell lines. R948K showed a slight increase in activity, which was not statistically significant. R948K is a conservative substitution and may be a natural polymorphism with no oncogenic consequences.
      HER3 Glu-909 participates in the heterodimer interface (Figs. 2B and 5), and MD simulation suggested Glu-909 forms a salt bridge with HER2 Lys-716 (Fig. 6A). HER3 E909G has recently been identified as an oncogenic mutation in gastric and breast cancers, showing increased Akt and ERK signaling in the presence of HER2 (
      • Jaiswal B.S.
      • Kljavin N.M.
      • Stawiski E.W.
      • Chan E.
      • Parikh C.
      • Durinck S.
      • Chaudhuri S.
      • Pujara K.
      • Guillory J.
      • Edgar K.A.
      • Janakiraman V.
      • Scholz R.-P.
      • Bowman K.K.
      • Lorenzo M.
      • Li H.
      • Wu J.
      • Yuan W.
      • Peters B.A.
      • Kan Z.
      • Stinson J.
      • Mak M.
      • Modrusan Z.
      • Eigenbrot C.
      • Firestein R.
      • Stern H.M.
      • Rajalingam K.
      • Schaefer G.
      • Merchant M.A.
      • Sliwkowski M.X.
      • de Sauvage F.J.
      • Seshagiri S.
      Oncogenic ERBB3 mutations in human cancers.
      ). We created HER3 mutants E909K, E909A, and E909G to assess how changes at this location affected the specific activity of the HER2-HER3 dimer. Interestingly, all three mutants displayed increased activation of HER2 relative to wild type HER3, with E909G showing the greatest effect (Fig. 7B). Reversing the charge of Glu-909 by mutating to lysine would disrupt the HER3 Glu-909-HER2 Lys-716 salt bridge, but it allows the formation of other ionic interactions with the neighboring HER2 Glu-717. In contrast, mutation of HER3 Glu-909 to alanine or glycine disrupts this charge interaction and allows the dimerization interface to shift toward the HER2 αC-helix, favoring increased activation of HER2. These results suggest that Glu-909 is an important modulator of activity at the HER2-HER3 dimer interface and that mutations at that site are activating through an allosteric mechanism.
      In conclusion, this study extends our understanding of the structural interactions that contribute to the HER2-HER3 asymmetric dimer using both direct measurements by carboxyl group footprinting MS and MD simulations. We have also demonstrated that HER2 activation can occur independent of tyrosine phosphorylation on its activation loop. Given the importance of HER2-HER3 interaction in several cancer types and the clinical interest in mutations of HER2 and other members of the ErbB family (
      • Bose R.
      • Kavuri S.M.
      • Searleman A.C.
      • Shen W.
      • Shen D.
      • Koboldt D.C.
      • Monsey J.
      • Goel N.
      • Aronson A.B.
      • Li S.
      • Ma C.X.
      • Ding L.
      • Mardis E.R.
      • Ellis M.J.
      Activating HER2 mutations in HER2 gene amplification negative breast cancer.
      ), the ability to rapidly evaluate and understand their consequences is critical. This study provides valuable insights into ErbB family kinase structure and interactions, which can guide the design of future therapies.

      Acknowledgments

      We thank Michael Gross, Henry Rohrs, Ilan Vidavsky, Leslie Hicks, and Sophie Alvarez for their mass spectrometry support and Brian Gau for programming. We also thank Linda Pike, Carl Frieden, Paul Schlessinger, and Dan Leahy for valuable discussions and advice.

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