jbc.m113.474882 key interactions at the her2-her3 dimer interface · 2013-07-10 · key...

Key Interactions at the HER2-HER3 Dimer Interface

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

Timothy S. Collier1, Karthikeyan Diraviyam3, John Monsey1, Wei Shen1, David Sept3 and Ron

Bose1,2

1Division of Oncology, Department of Medicine and 2Department of Cell Biology and Physiology,

Washington University School of Medicine, St. Louis, MO 63110.

3Department of Biomedical Engineering and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109.

*Running title: Key Interactions at the HER2-HER3 Kinase Dimer Interface

To whom correspondence should be addressed: Ron Bose, M.D., Ph.D., Division of Oncology, Department of Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, Campus Box 8069, 660 S. Euclid Ave., St. Louis, MO, USA 63110. Tel.: (314) 747-9308; Fax: (314) 747-9308; E-mail: [email protected] Keywords: receptor tyrosine kinase, mass spectrometry (MS), protein-protein interactions, Molecular dynamics, Epidermal growth factor receptor (EGFR) 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. SUMMARY

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 EGFR/HER/ErbB family and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Prior 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 (MS), 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 (A-loop), as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding, but did not require A-loop phosphorylation at Y877. Molecular dynamics (MD) 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 K716-HER3 E909, HER2 E717-HER3 K907, and HER2 D871-HER3 R948 were identified by MD. We also evaluate the effect of the cancer associated mutations HER2 D769H/Y, 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 EGFR/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.474882The latest version is at JBC Papers in Press. Published on July 10, 2013 as Manuscript M113.474882

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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The epidermal growth factor receptor (EGFR)/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 (1, 2). 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 (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 (4).

HER2 is overexpressed in 20-30% of breast cancers (5) and is the preferred heterodimerization partner of all other ErbB family members, especially HER3 (6–8). 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 (1, 9–12). To date, the structure of an ErbB family kinase heterodimer has yet to be solved. While 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)(13), and hydroxyl radical labeling (14). H/DX labels amide hydrogens while hydroxyl radical labeling modifies aromatic, aliphatic, and sulfur-containing sidechains. 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 (15–18). These techniques suffer some limitations when investigating membrane-associated proteins, such as extensive back-exchange (in the case of H/DX) or non-specific 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 (19, 20).

An alternative to HD/X and hydroxyl radical footprinting is labeling carboxylic acid sidechains (Glu and Asp) with the 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC)-mediated incorporation of glycine ethyl ester (GEE)(21, 22). 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 (23).

In this article, 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 occurs 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 Ni-chelating liposome, increasing the local concentration and facilitating protein-protein interaction (1, 24). 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


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either ATP or its non-hydrolyzable analogue, AMP-PNP. We observed similar activation loop conformational changes in the non-receptor 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. EXPERIMENTAL PROCEDURES

Protein Expression and Liposome Preparation-HER2 and HER3 kinase domains were expressed as previously described (24). Briefly, HER2 kinase domain (residues 704-1029) and HER3 kinase domain (residues 675-1001) were cloned into pFastBacHT vector. Site-directed mutagenesis was performed using the QuikChange II method (Agilent) and constructs were confirmed by DNA sequence analysis. Recombinant baculovirus was made following the Bac-to-Bac protocol (Invitrogen). Spodoptera frugiperda Sf9 cells were infected with baculovirus at one multiplicity of infection and harvested 48 hours post-infection. His6-tagged HER2 kinase-domain was purified using Ni-NTA beads (Qiagen). His6-tagged HER3 kinase-domain was purified using Ni-NTA beads followed by gel-filtration chromatography. Numbering of HER2 and HER3 residues is based on their crystal structures (3PP0 and 3LMG, respectively). Liposomes were prepared as previously described (24) using the reverse phase method detailed by Szoka et al (25) followed by extrusion through 200 nm polycarbonate membranes in a mini-extruder (Avanti Polar Lipids, Alabaster, AL) to generate small unilamellar vesicles (SUV) with diameters measured by dynamic light scattering of 180-190 nm. Nickel-liposomes were SUV’s containing 95 mol% DOPC and 5 mol% Ni-NTA-DOGS. Recombinant full-length Src was purchased (Millipore).

Protein Dimerization and Phosphorylation-Proteins were kept in stock solutions containing 20 mM Tris-HCl, 150 mM

NaCl, and 1mM dithiothreitol (DTT) (pH 8.0). The HER2 stock solution also contained 0.01% Tween-20. Dimerization experiments were performed by equilibrating HER2 and HER3 in a 1:1 ratio with nickel liposomes on ice for 15 min. Protein phosphorylation was initiated by adding ATP and MgCl2 to the dimer solution (final concentration 100 µM ATP and 10 mM MgCl2). Evaluation in the presence of non-hydrolysable ATP mimetic was performed similarly, but with 100 µM AMP-PNP instead of ATP. Sample were then subjected to carboxyl group footprinting after incubating 5 minutes on ice after addition of ATP and MgCl2.

Carboxyl Group Footprinting Reaction and GeLC Mass Spectrometry-Labeling reactions and subsequent LC-MS/MS analyses were performed in triplicate for all conditions. Protein samples (10 µL of 2.5 µM) in 20 mM Tris-HCl, 100 mM NaCl, pH 7.5 was combined with 0.5 µL of 2M GEE. The reaction was initiated with the addition of 0.5 µL of 50 mM EDC. Reactions were quenched after 10 minutes with the addition of 11 µL of 1 M ammonium acetate. Immediately after quenching the reaction, 5X SDS-PAGE sample-loading buffer, containing DTT at the reducing agent, was mixed with each sample and the mixture boiled for 5 min. The samples were loaded onto a 10% SDS-PAGE gel and detected by SimplyBlue SafeStain (Invitrogen). In-gel digestion was performed as per Shevchenko et al (26). Tryptic peptides were separated by a reverse-phase Eksigent 1D+ nano-liquid chromatography system (Eksigent) directly coupled to an Orbitrap Velos-Pro mass spectrometer (Thermo Fisher Scientific). The resulting RAW files were subjected to chromatographic alignment, peak quantification, and peak list generation using Progenesis LC-MS (Nonlinear Dynamics). Peptides were identified in MASCOT server 2.3 (Matrix Science) using a custom database containing Src in addition to the HER2 and HER3 kinase domain construct sequences and a list of common contaminants added to the E. coli K12 database (4,280 total sequences) (27). Cysteine carbamidomethylation was considered as a fixed modification, while custom-built GEE modifications (+57.021464 Da and +85.052764 Da specific to glutamic and aspartic acid), methionine oxidation, and phosphorylation of serine, threonine, and tyrosine


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residues were all considered as variable modifications. Other considerations for the MASCOT search include: enzyme, trypsin; maximum number of missed cleavage, 2; peptide mass tolerance = 10 ppm; fragment ion tolerance 0.8 Da; instrument type, ESI-trap. Residue level quantification was achieved using Progenesis LC-MS to quantify feature extracted ion chromatograms in addition to an Excel-Based Visual Basic Application described in reference (28).

Antibodies and Western Blotting-Blot for Src phosphorylation was performed by incubation in 1:2,000 dilution of 4G10 anti-pTyr antibody (Upstate Biotechnology, Lake Placid, NY) followed by 1:10,000 dilution of ECL peroxidase labeled anti-mouse antibody (Amersham Biosciences, Piscataway, NJ). Data shown is representative of at least two experiments.

Homology modeling and Molecular Dynamics-A HER2-HER3 heterodimeric structural model was built using Modeller (29). Initially individual models for HER2 monomer were built with 3PP0 x-ray structure as the template and HER3 monomer with 3LMG as the template. The heterodimer was then built by replacing the HER2 donor kinase in the HER2 homodimer model with HER3. This is used as the starting structure for Molecular Dynamics simulations. A total of three simulations each of 100ns duration were carried out for both the heterodimer and individual monomers. Simulations were carried out using NAMD (30, 31) in explicit water under standard room temperature (300K) and pressure (1atm) conditions in an NPT ensemble. AMBER (32) forcefield was used for the simulations. SASA and H-bond analysis were carried out for the last 25ns of simulations using VMD (33). Kinase Assays - Radiometric kinase assays were performed as per ref. (24). Briefly, wild-type and mutant HER2 and HER3 constructs were evaluated in solution (monomers) or when attached to Ni-liposomes to induce dimerization. Assays were performed with 100 µM of the HER2 optimized peptide substrate biotin-GGMEDIYFEFMGGKKK (34) in 30 mM Tris, pH 7.5, 10 mM MgCl2, 100 µM Na3VO4, 2 mM DTT, 100 µM ATP, 1 µCi of [γ-32P]ATP, 60 mM NaCl, 5% glycerol, 0.005% Tween-20 in 25 µL total reaction volume. The reactions were initiated

with the addition of 1.25 µM kinase, allowed to react for 8 min at 30 °C and quenched with EDTA. Avidin (100 µg) was added, and samples were transferred to centrifugal filtration units with 30,000 molecular weight cut-off (Millipore). Samples were washed three times with 0.5 M sodium phosphate, 0.5 M NaCl, pH 8.5. Retained 32P was measured by scintillation counting. For the evaluation of kinase activity on liposomes, kinases were incubated on liposomes on ice for 15 min before initiating the reaction. The total lipid concentration was 0.5 mg/mL. For heterodimerization reactions, kinases were mixed prior to the addition of liposomes. Statistical comparisons were performed using the Student’s t-test and results are representative of at least two independent experiments. RESULTS

Carboxyl Group Footprinting Mass Spectrometry-We recombinantly expressed and purified His6-tagged HER2 and HER3 kinase domains and formed dimers by binding them to liposomes containing 5 mol% nickel chelating lipid (Ni-NTA-DOGS). We performed carboxyl group footprinting experiments on four conditions – kinase monomers in solution, kinase heterodimers on liposome, heterodimers on liposome with the addition of ATP, and heterodimers on liposome with the addition of the non-hydrolyzable ATP analogue, AMP-PNP – and examined the relative extents of modification of aspartic and glutamic acid residues with the EDC mediated addition of glycine ethyl ester (GEE) (Table S1). Carboxyl group footprinting using GEE labeling produces a mass shift of 85 Da (or 57 Da after ester hydrolysis) that can be localized and quantified using LC-MS/MS (Fig. 1).

Footprinting between HER2 and HER3 Monomers and Heterodimer - The asymmetric dimer model for ErbB family kinase activation postulates that specific residues on the C-terminal lobe of one kinase monomer contact N-terminal lobe residues of another monomer (1). In this arrangement, the former kinase monomer is often referred to as the “donor” kinase and the latter as the “acceptor”. Given the divergence of the HER3 kinase domain’s N-lobe relative to the rest of the ErbB family, it is predicted to be a poor acceptor monomer (1, 7, 9). However, HER3 retains significant homology in its C-lobe with the rest of


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the ErbB family and can therefore serve as a donor monomer in heterodimers. We combined equimolar amounts of HER2 and HER3 in the presence of liposomes that facilitate dimer formation and compared the extents of carboxyl group footprinting to the monomers in solution. Decreased levels of GEE labeling were observed on several residues at the HER2 acceptor interface (E717 and E719) and αC-helix (E766, D769, and E770) (Fig. 2A) and additionally at the HER3 donor interface (D903, E906, E909, D920, and D932) (Fig. 2B). Using previously published crystal structures of HER2 and HER3 kinase domains, we generated a three-dimensional model of a HER2-HER3 asymmetric heterodimer. Specifically, the C-lobe region of HER3 kinase domain structure (Protein Data Bank (PDB) code:3LMG) was aligned to the C-lobe region of the donor monomer in the HER2 kinase domain homodimer structure (PDB: 3PP0) based on the significant homology between these domains across all EGFR/ErbB family members (9, 10). This model, shown in Fig. 2C, highlights GEE labeled acidic residues colored based on their extents of modification when dimerized relative to monomers in solution. The carboxyl group footprinting MS results support the model as evidenced by residues having decreased GEE labeling (highlighted in red) being located at the dimer interface. We do not detect changes in GEE labeling on HER2 donor interface residues (Fig. 2A; E936, E939, E992 and D993) or at the HER3 acceptor interface (Fig. 2B; E687 and E689). Based on the analytical reproducibility, the threshold of detection is approximately 10%. Therefore, if HER2 or HER3 homodimers or heterodimers in the opposite configuration were present, they are minor species representing less than 10% of the population. The HER2-HER3 heterodimer with HER3 as donor and HER2 as acceptor is the major species, accounting for greater than 90% of the dimer population.

Activation Loop Conformation Changes in Presence of Nucleotide-Differential extents of GEE labeling were also observed for acidic residue sidechains that are not implicated in inter-molecular interactions. The activation loop consists of residues 863-884 in HER2 and 833-854 in HER3, each beginning with the highly conserved DFG motif and extending to the WMALE motif. As shown in Fig. 3A HER2

D863 in the DFG motif exhibited increased GEE labeling compared to its monomeric state when dimerized on the Ni-liposomes, and this increased further after the addition of ATP or the non-hydrolyzable ATP-analogue, AMP-PNP. Although no significant change was detected with dimerization alone, the five acidic residues (D871, D873, E874, E876, and D880) along the activation loop of HER2 showed increased GEE labeling when dimerized on the Ni-liposomes and incubated with ATP or AMP-PNP (Fig. 3A). In contrast, the DFG aspartate in HER3 (D833) and other acidic residues along the HER3 A-loop (D838, D843, D844, and E851), did not show any statistically significant changes in GEE labeling (Fig. 3B). The relative levels of autophosphorylation were also measured for HER2 and HER3 in each experimental condition. High levels of phosphorylation were only detected for HER2 Y877 when in the presence of ATP (Fig. 3C), but the increases in GEE labeling of the HER2 A-loop occurred with either ATP of AMP-PNP addition. These results suggest that the HER2 A-loop adopts an open conformation (Fig. 3D) independently of Y877 phosphorylation, but with the requirement for both dimerization and nucleotide binding.

Footprinting of Src Activation Loop-In order to ensure that the A-loop conformational changes observed in HER2 kinase domain were in fact the result of adopting open vs. closed conformations (Fig. 3D), we also performed carboxyl group footprinting experiments on the cytoplasmic tyrosine kinase Src. Src is not dependent on dimerization for activation and so could be examined with the addition of ATP, AMP-PNP or buffer without the need for liposomes. As indicated in Fig. 4, we saw a similar increase in GEE labeling of acidic sidechains in the Src A-loop with the addition of ATP, correlating with detection of increased phosphorylation of the A-loop autophosphorylation site (Y416) by mass spectrometry and western blot (Fig. 4B). However, the same increase in GEE labeling of A-loop residues did not occur when Src was incubated with AMP-PNP (Fig. 4A). These results strongly suggest that the GEE labeling changes of A-loop residues in both HER2 and Src are the result of adopting an open A-loop conformation. Previous studies have shown that


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Src adopts an open A-loop conformation in a phosphorylation-dependent manner(35–37).

Molecular Dynamics of HER2-HER3 Asymmetric Heterodimer-To investigate what interactions may play a role in dimerization and stabilize the open conformation of the A-loop, we subjected our 3D model of the HER2-HER3 kinase domain heterodimer (Fig. 2C) and their respective kinase domain monomer structures to molecular dynamics simulations. Homology modeling of HER2-HER3 heterodimer was accomplished using the HER2 homodimer crystal structure as template. 100 ns simulations of the heterodimer and individual monomers were performed using NAMD with AMBER forcefield parameters. A probe radius of 1.4 Å was used to calculate the solvent accessible surface area (SASA) for selected acidic residues corresponding to those that were identified using carboxyl group footprinting MS. The analysis was performed on 50 snapshots corresponding to the last 25 ns of the 100 ns simulation for both dimer and monomer model systems.

A comparison of the SASA change between the monomer and heterodimer and the GEE footprinting measurement of the same residues is illustrated in Fig. 5. Most of the changes in SASA for residues corresponding to the HER2 acceptor interface (E717, E719), αC-helix (E766, D769), and the donor interface (E936, E992, E993) agree well with the GEE footprinting analysis (Fig. 5A). In the case of E744, the residue is predominantly solvent accessible in the simulated heterodimer, but in monomer simulations the residue is involved in partial interactions with K777. A similar rationale causes the discrepancy with respect to simulation of SASA changes with E975. Likewise, we see strong agreement of SASA calculations from the MD simulation with the carboxyl group footprinting MS data for HER3 (Fig. 5B). There are no significant changes in SASA at the HER3 acceptor interface (E689, E712, E714, D739), while decreases in SASA are observed at the HER3 donor interface (D903, 909, D920). The MD simulation shows that D932 is near the HER3 donor interface, but is still solvent accessible in the heterodimer. Differences between the MD and chemical footprinting measurements may result from the lack of the lipid surface and the His-tag construct in silico or from the difference in the

time scale of the simulation (100 ns) versus the duration of the carboxyl group footprinting experiment (minutes). However, despite these differences, the changes in SASA observed by MD agrees well with the carboxyl group footprinting results.

The MD simulation on the HER2-HER3 heterodimer identified several persistent H-bonds (Table 1, Fig. 6). At the HER2-HER3 dimerization interface, there are two persistent interactions including HER2-K716 to HER3-E909 (Fig. 6A) in addition to HER2-E717 to HER3-K907. We also observe several intramolecular salt bridges and hydrogen bonds involved in coordination of the catalytic loop with the γ-phosphate of ATP (Fig. 6B and 6C) as well as stabilize the open conformation of the unphosphorylated HER2 activation loop, however these interaction are more dynamic and variable, involving D769 and Y772 and D871 on Her2 and D920, R948 and R951 on HER3 (Fig 6D).

Functional Analysis of Footprinted Residues and Known Cancer Associated Mutations – We evaluated the effects of residues involved in HER2-HER3 dimerization. These analyses utilized in vitro kinase assays in which recombinant His6-tagged wild type or mutant HER2 and HER3 constructs were assayed as monomers or attached to Ni-liposomes to induce dimer formation. Further, several interface residues have known cancer associated mutations including HER2 D769H and D769Y, HER3 E909G, and HER3 R948K (38–41).

We measured kinase activity of HER2 wild type, D769A, D769H and D769Y (Fig. 7A). As we have previously shown, the attachment of the HER2 and HER3 kinase domains to nickel liposomes resulted in increased specific activity relative to the kinases in solution alone (24). The specific activity of HER2 D769A was greatly reduced both as monomers and heterodimers with HER3 WT. HER2 D769H and D769Y showed 3-fold more specific activity in heterodimers, in addition to increased activity as monomers.

We also assessed the kinase activity of mutations of two HER3 residues relative to wild type. HER3 E909 was determined to participate in the dimer interface (Fig 2B). We assessed the activity of several mutations of these sidechains including E909G, A, and K (Fig. 7B). E909G, also numbered E928G when including the signal


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peptide, has been identified as a cancer associated mutation(42–45). Wild type HER3 kinase domain showed a 5.7 fold increase in kinase activity when dimerized with HER2 relative to HER2 monomer activity. To determine whether the acidic sidechain was necessary for activation, we assessed the activity of HER3 E909K, which resulted in increased activation of HER2 relative to HER3 wild type, a 10-fold increase over HER2 monomer activity. HER3 E909A showed a further increase in HER2 activation (20-fold), while E909G showed the greatest increase with a 32-fold increase in HER2 activation.

In the molecular dynamics simulation, HER3 R948 was implicated in the intermolecular stabilization of the HER2 activation loop (Fig. 6D). An R948K mutation has likewise been observed in cancer cell lines derived from many different tissues (44). HER3 R948K gave a marginal increase in activation of HER2 (6.8 fold relative to HER2 in solution)(Fig. 7C) relative to HER3 WT. However, the increase was not statistically significant. Eliminating the basic sidechain with a R948A mutation produced a modest yet statistically significant decrease in HER2 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 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 inter-molecular interactions that provide additional stability to the open, non-phosphorylated 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 (6–8). 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 E717 and HER3 E909, which in MD simulations are involved in salt-bridge interaction with HER3 K907 and HER2 K716, respectively (Fig. 6). Analogous salt-bridge interactions were previously observed in HER4 homodimers (23). Hydrophobic interactions also play a key role at the dimerization interface and we will further explore those interactions in the future (46).

The activation loop (A-loop) regulates access of target substrates to the catalytic loop, which facilitates phosphoryl transfer. Upon kinase activation, the A-loop undergoes a dramatic conformation shift in which it extends to uncover the kinase substrate-binding pocket (Fig. 3D)(36, 47). 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 (48). The study of the Src-family kinase Lck crystal structure, in which Y394 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 (49). However, the requirement for A-loop tyrosine phosphorylation in EGFR and HER2 is still controversial (50–55). 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 (46). The presence of several


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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 non-hydrolyzable ATP analogue. We thus conclude that the open A-loop conformation in HER2 is dependent on dimerization and nucleotide binding, but 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 E766-R868, and interactions involving K753 and D863 with the coordination with Mg2+ and ATP in the catalytic pocket have been previously described and also appear in our simulations (1, 56). We also observe H-bond interactions involving D845, R849, and N850 in the catalytic loop (C-loop) that have been implicated in coordination and transfer of the γ-phosphate of ATP (Fig. 6C) (46, 56, 57). MD simulations also revealed novel interactions that stabilize the unphosphorylated A-loop of HER2. A persistent backbone-backbone interaction between Y877 and F899 is likely to be further stabilized by the parallel-displaced π-stacking of the two aromatic sidechains, and interaction not accounted for by the simulation that would provide 0.5 to 1.0 kcal/mol more stability (Fig. 6E) (58). We found interactions between several groups of residues, D769, Y772 and D871 on HER2 with D920, R948 and R951 on HER3, provide a possible mechanism for inter-molecular stabilization of the open and extended activation loop (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 (38–40). We recently reported that D769H/Y were activating mutations in breast cancer and possessed greater specific activity when assayed as HER2 homodimers (38). 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). Further, D769A results in a loss of kinase activity. Potential interpretations of these results include increased hydrophobic interaction between HER2 D769H/Y and HER3 I919 and M923, or intramolecular interactions with the HER2 A-loop. Our MD simulations show the interaction of D769 with the amide proton of HER2 L869 in the A-loop, providing additional stability to the open conformation. Mutation to histidine or tyrosine increases the sidechain length and hydrophobicity, which could provide additional stability to the open activation loop through interactions with the HER2 L869 sidechain. This additional stability accounts for the observed increases in D769H/Y monomer and dimer activity relative to wild type (Fig. 7A). In contrast, replacing the aspartate with a shorter, non-polar, 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 R948 with HER2 D871 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 to 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.


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HER3 E909 participates in the heterodimer interface (Fig. 2B and 5) and MD simulation suggested E909 forms a salt bridge with HER2 K716 (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 (45). 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 E909 by mutating to lysine would disrupt the HER3 E909-HER2 K716 salt bridge, but allows the formation of other ionic interactions with the neighboring HER2 E717. In contrast, mutation of HER3 E909 to alanine or glycine disrupts this charge interaction and allows the dimerization interface to shift towards the HER2 αC-helix, favoring increased activation of HER2. These results

suggest that E909 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 (38), 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.


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REFERENCES

1. Zhang, X., Gureasko, J., Shen, K., Cole, P. A., and Kuriyan, J. (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137–49

2. Arkhipov, A., Shan, Y., Das, R., Endres, N. F., Eastwood, M. P., Wemmer, D. E., Kuriyan, J., and Shaw, D. E. (2013) Architecture and membrane interactions of the EGF receptor. Cell 152, 557–69

3. Citri, A., Skaria, B. S., and Yarden, Y. (2003) The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp Cell Res 284, 54–65

4. Holbro, T., Beerli, R. R., Maurer, F., Koziczak, M., Barbas, C. F., and Hynes, N. E. (2003) The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A 100, 8933–8

5. Slamon, D., Clark, G., Wong, S., Levin, W., Ullrich, A., and McGuire, W. (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177–182

6. Tzahar, E., and Waterman, H. (1996) A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 16, 5276

7. Graus-Porta, D., Beerli, R. R., Daly, J. M., and Hynes, N. E. (1997) ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16, 1647–55

8. Karunagaran, D., Tzahar, E., Beerli, R. R., Chen, X., Graus-Porta, D., Ratzkin, B. J., Seger, R., Hynes, N. E., and Yarden, Y. (1996) ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 15, 254–64

9. Aertgeerts, K., Skene, R., Yano, J., Sang, B.-C., Zou, H., Snell, G., Jennings, A., Iwamoto, K., Habuka, N., Hirokawa, A., Ishikawa, T., Tanaka, T., Miki, H., Ohta, Y., and Sogabe, S. (2011) Structural analysis of the mechanism of inhibition and allosteric activation of the kinase domain of HER2 protein. J Biol Chem 286, 18756–65

10. Shi, F., Telesco, S. E., Liu, Y., Radhakrishnan, R., and Lemmon, M. a (2010) ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation. Proc Natl Acad Sci U S A 107, 7692–7

11. Qiu, C., Tarrant, M. K., Choi, S. H., Sathyamurthy, A., Bose, R., Banjade, S., Pal, A., Bornmann, W. G., Lemmon, M. a, Cole, P. a, and Leahy, D. J. (2008) Mechanism of activation and inhibition of the HER4/ErbB4 kinase. Structure 16, 460–7

12. Jura, N., Shan, Y., Cao, X., Shaw, D. E., and Kuriyan, J. (2009) Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3. Proc Natl Acad Sci U S A 106, 21608–13

13. Wales, T. E., and Engen, J. R. (2006) Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev 25, 158–70

14. Xu, G., and Chance, M. R. (2007) Hydroxyl radical-mediated modification of proteins as probes for structural proteomics. Chem Rev 107, 3514–43

15. Konermann, L., Tong, X., and Pan, Y. (2008) Protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches. J Mass Spectrom 43, 1021–36


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


11

16. Roulhac, P. L., Powell, K. D., Dhungana, S., Weaver, K. D., Mietzner, T. A., Crumbliss, A. L., and Fitzgerald, M. C. (2004) 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. Biochemistry 43, 15767–74

17. Zhang, J., Adrián, F. J., Jahnke, W., Cowan-Jacob, S. W., Li, A. G., Iacob, R. E., Sim, T., Powers, J., Dierks, C., Sun, F., Guo, G.-R., Ding, Q., Okram, B., Choi, Y., Wojciechowski, A., Deng, X., Liu, G., Fendrich, G., Strauss, A., Vajpai, N., Grzesiek, S., Tuntland, T., Liu, Y., Bursulaya, B., Azam, M., Manley, P. W., Engen, J. R., Daley, G. Q., Warmuth, M., and Gray, N. S. (2010) Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors. Nature 463, 501–6

18. Chitta, R. K., Rempel, D. L., Grayson, M. a, Remsen, E. E., and Gross, M. L. (2006) Application of SIMSTEX to oligomerization of insulin analogs and mutants. J Am Soc Mass Spectrom 17, 1526–34

19. Hebling, C. M., Morgan, C. R., Stafford, D. W., Jorgenson, J. W., Rand, K. D., and Engen, J. R. (2010) Conformational Analysis of Membrane Proteins in Phospholipid Bilayer Nanodiscs by Hydrogen Exchange Mass Spectrometry. Anal Chem 82, 5415–5419

20. Morgan, C. R., Hebling, C. M., Rand, K. D., Stafford, D. W., Jorgenson, J. W., and Engen, J. R. (2011) Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs. Mol Cell Proteomics 10, M111.010876

21. Hoare, D. G., Olson, A., and Koshland, D. E. (1968) The reaction of hydroxamic acids with water-soluble carbodiimides. A Lossen rearrangement. J Am Chem Soc 90, 1638–43

22. Hoare, D. G., and Koshland, D. E. (1967) A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J Biol Chem 242, 2447–53

23. Zhang, H., Shen, W., Rempel, D., Monsey, J., Vidavsky, I., Gross, M. L., and Bose, R. (2011) Carboxyl-group footprinting maps the dimerization interface and phosphorylation-induced conformational changes of a membrane-associated tyrosine kinase. Mol Cell Proteomics 10, M110.005678

24. Monsey, J., Shen, W., Schlesinger, P., and Bose, R. (2010) Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system. J Biol Chem 285, 7035–44

25. Szoka, F., Olson, F., Heath, T., Vail, W., Mayhew, E., and Papahadjopoulos, D. (1980) Preparation of unilamellar liposomes of intermediate size (0.1–0.2 µm) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes. Biochim Biophys Acta - Biomembranes 601, 559–571

26. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V, and Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1, 2856–60

27. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–67

28. Gau, B. C., Chen, J., and Gross, M. L. (2013) Fast photochemical oxidation of proteins for comparing solvent-accessibility changes accompanying protein folding: Data processing and application to barstar. Biochimica et biophysica acta 1834, 1230–8

29. Fiser, A., and Sali, A. (2003) Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol 374, 461–91


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


12

30. Nelson, M. T., Humphrey, W., Gursoy, A., Dalke, A., Kale, L. V., Skeel, R. D., and Schulten, K. (1996) NAMD: a Parallel, Object-Oriented Molecular Dynamics Program. Int J High Perform Comput Appl 10, 251–268

31. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kalé, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26, 1781–802

32. Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. (1995) A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules. J Am Chem Soc 117, 5179–5197

33. Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visual molecular dynamics. J Mol Graph 14, 33–8, 27–8

34. Jan, A. Y., Johnson, E. F., Diamonti, A. J., Carraway III, K. L., and Anderson, K. S. (2000) Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis. Biochemistry 39, 9786–803

35. Roskoski, R. (2004) Src protein-tyrosine kinase structure and regulation. Biochem Biophys Res Commun 324, 1155–64

36. Huse, M., and Kuriyan, J. (2002) The conformational plasticity of protein kinases. Cell 109, 275–82

37. Sun, G., Ramdas, L., Wang, W., Vinci, J., McMurray, J., and Budde, R. J. A. (2002) Effect of autophosphorylation on the catalytic and regulatory properties of protein tyrosine kinase Src. Arch Biochem Biophys 397, 11–7

38. 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., and Ellis, M. J. (2013) Activating HER2 Mutations in HER2 Gene Amplification Negative Breast Cancer. Cancer Discov 3, 224–37

39. 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., and Lee, S. H. (2006) ERBB2 kinase domain mutation in the lung squamous cell carcinoma. Cancer Lett 237, 89–94

40. Lee, J. W., Soung, Y. H., Seo, S. H., Kim, S. Y., Park, C. H., Wang, Y. P., Park, K., Nam, S. W., Park, W. S., Kim, S. H., Lee, J. Y., Yoo, N. J., and Lee, S. H. (2006) Somatic mutations of ERBB2 kinase domain in gastric, colorectal, and breast carcinomas. Clin Canc Res 12, 57–61

41. The Cancer Genome Atlas Network (2012) Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70

42. Kan, Z., Jaiswal, B. S., Stinson, J., Janakiraman, V., Bhatt, D., Stern, H. M., Yue, P., Haverty, P. M., Bourgon, R., Zheng, J., Moorhead, M., Chaudhuri, S., Tomsho, L. P., Peters, B. A., Pujara, K., Cordes, S., Davis, D. P., Carlton, V. E. H., Yuan, W., Li, L., Wang, W., Eigenbrot, C., Kaminker, J. S., Eberhard, D. A., Waring, P., Schuster, S. C., Modrusan, Z., Zhang, Z., Stokoe, D., De Sauvage, F. J., Faham, M., and Seshagiri, S. (2010) Diverse somatic mutation patterns and pathway alterations in human cancers. Nature 466, 869–73

43. Banerji, S., Cibulskis, K., Rangel-Escareno, C., Brown, K. K., Carter, S. L., Frederick, A. M., Lawrence, M. S., Sivachenko, A. Y., Sougnez, C., Zou, L., Cortes, M. L., Fernandez-Lopez, J. C., Peng, S., Ardlie, K. G., Auclair, D., Bautista-Piña, V., Duke, F., Francis, J., Jung, J., Maffuz-Aziz, A., Onofrio, R. C., Parkin, M., Pho, N. H., Quintanar-Jurado, V., Ramos, A. H., Rebollar-Vega, R., Rodriguez-Cuevas, S., Romero-Cordoba, S. L., Schumacher, S. E., Stransky, N., Thompson, K. M., Uribe-Figueroa, L., Baselga, J., Beroukhim, R., Polyak, K., Sgroi, D. C., Richardson, A. L., Jimenez-Sanchez, G., Lander, E. S., Gabriel, S. B., Garraway, L. A., Golub, T. R., Melendez-Zajgla, J., Toker, A., Getz, G., Hidalgo-Miranda, A., and Meyerson, M. (2012) Sequence analysis of mutations and translocations across breast cancer subtypes. Nature 486, 405–9


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


13

44. Barretina, J., Caponigro, G., Stransky, N., Venkatesan, K., Margolin, A. A., Kim, S., Wilson, C. J., Lehár, J., Kryukov, G. V, Sonkin, D., Reddy, A., Liu, M., Murray, L., Berger, M. F., Monahan, J. E., Morais, P., Meltzer, J., Korejwa, A., Jané-Valbuena, J., Mapa, F. A., Thibault, J., Bric-Furlong, E., Raman, P., Shipway, A., Engels, I. H., Cheng, J., Yu, G. K., Yu, J., Aspesi, P., De Silva, M., Jagtap, K., Jones, M. D., Wang, L., Hatton, C., Palescandolo, E., Gupta, S., Mahan, S., Sougnez, C., Onofrio, R. C., Liefeld, T., MacConaill, L., Winckler, W., Reich, M., Li, N., Mesirov, J. P., Gabriel, S. B., Getz, G., Ardlie, K., Chan, V., Myer, V. E., Weber, B. L., Porter, J., Warmuth, M., Finan, P., Harris, J. L., Meyerson, M., Golub, T. R., Morrissey, M. P., Sellers, W. R., Schlegel, R., and Garraway, L. A. (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–7

45. 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., and Seshagiri, S. (2013) Oncogenic ERBB3 Mutations in Human Cancers. Cancer cell 23, 603–17

46. Telesco, S. E., and Radhakrishnan, R. (2009) Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study. Biophys J 96, 2321–34

47. Stamos, J., Sliwkowski, M. X., and Eigenbrot, C. (2002) Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J Biol Chem 277, 46265–72

48. Porter, M. (2000) Reciprocal Regulation of Hck Activity by Phosphorylation of Tyr527 and Tyr416. Effect of Introducing a High Affinity Intramolecular SH2 Ligand. J Biol Chem 275, 2721–2726

49. Yamaguchi, H., and Hendrickson, W. A. (1996) Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation. Nature 384, 484–9

50. Gotoh, N., Tojo, A., Hino, M., Yazaki, Y., and Shibuya, M. (1992) 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. Biochem Biophys Res Commun 186, 768–774

51. Biscardi, J. S. (1999) c-Src-mediated Phosphorylation of the Epidermal Growth Factor Receptor on Tyr845 and Tyr1101 Is Associated with Modulation of Receptor Function. J Biol Chem 274, 8335–8343

52. Zhang, H. T., O’Rourke, D. M., Zhao, H., Murali, R., Mikami, Y., Davis, J. G., Greene, M. I., and Qian, X. (1998) Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential. Oncogene 16, 2835–42

53. Segatto, O., Lonardo, F., Pierce, J. H., Bottaro, D. P., and Di Fiore, P. P. (1990) The role of autophosphorylation in modulation of erbB-2 transforming function. New Biol 2, 187–95

54. Xu, W., Yuan, X., Beebe, K., Xiang, Z., and Neckers, L. (2007) Loss of Hsp90 association up-regulates Src-dependent ErbB2 activity. Mol Cell Biol 27, 220–8

55. Bose, R., Molina, H., Patterson, A. S., Bitok, J. K., Periaswamy, B., Bader, J. S., Pandey, A., and Cole, P. A. (2006) Phosphoproteomic analysis of Her2/neu signaling and inhibition. Proc Natl Acad Sci U S A 103, 9773–8

56. Parang, K., Till, J. H., Ablooglu, A. J., Kohanski, R. A., Hubbard, S. R., and Cole, P. A. (2001) Mechanism-based design of a protein kinase inhibitor. Nat Struct Biol 8, 37–41


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


14

57. Williamson, A. J. K., Smith, D. L., Blinco, D., Unwin, R. D., Pearson, S., Wilson, C., Miller, C., Lancashire, L., Lacaud, G., Kouskoff, V., and Whetton, A. D. (2008) Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis. Mol Cell Proteomics 7, 459–72

58. McGaughey, G. B. (1998) pi -Stacking Interactions. Alive and Well in Proteins. J Biol Chem 273, 15458–15463

Acknowledgements - The authors would like to 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.

FOOTNOTES *Research reported in this publication was supported by the National Institutes of Health under Award Number R01CA161001 (RB) and 8P41 GM103422-36 to Michael Gross and by the National Science Foundation NSF-DBI 0922879 to Leslie Hicks. TSC is supported by NIH T32 training grant 2T32HL007088-36. 1 To whom correspondence should be addressed: Ron Bose, M.D., Ph.D., Division of Oncology, Department of Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, Campus Box 8069, 660 S. Euclid Ave., St. Louis, MO, USA 63110. Tel.: (314) 747-9308; Fax: (314) 747-9308; E-mail: [email protected] 2The abbreviations used are: HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; EGFR, epidermal growth factor receptor; SASA, solvent accessible surface area; MS, mass spectrometry; MD, molecular dynamics; H-bond, hydrogen bond; A-loop, activation loop; GEE, glycine ethyl ester; EDC, 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide; ATP, adenosine triphosphate; AMP-PNP, adenylyl-imidodiphosphate FIGURE LEGENDS FIGURE 1. Mass Spectrometric detection, localization, and quantification of modified peptides. Unmodified and GEE-modified peptide LLDIDETEYHADGGK were detected in the same LC-MS run with high mass accuracy. The modified peptide’s tandem mass spectrum is shown in reflection to the unmodified peptide fragmentation spectrum, illustrating the 57 Da mass shift allowing identification of the peptide sequence and the localization of the GEE modification. Extents of modification are calculated based on the extracted ion chromatograms of modified and unmodified peptides. FIGURE 2. Carboxyl group footprinting MS reveals formation of HER2-HER3 Heterodimers. Relative extents of GEE labeling across four experimental conditions of acidic residues near the dimer interface of (A) HER2 and (B) HER3. (C) Three dimensional model of HER2-HER3 heterodimer assembled using existing crystal structures. GEE labeled residues are colored based on extents of modification during the formation of dimers in the presence of ATP relative to monomers in solution. FIGURE 3. Increases in HER2 activation loop GEE labeling with dimers in the presence of nucleotide. Relative extents of GEE labeling for acidic residues in the (A) HER2 and (B) HER3 activation loops. (C) Relative extent of phosphorylation of Y877 on the A-loop of HER2. (D) Structural Models showing inactive EGFR kinase domain (PDB: 2GS7) and active EGFR kinase domain (PDB: 2GS2) showing the closed and open conformations of the activation loop (highlighted in blue). The activation loop tyrosine (Y877 in HER2) is highlighted in pink. FIGURE 4. Carboxyl group footprinting of Src A-loop shows increased labeling correlates with Y416 phosphorylation. (A) GEE labeling measurements of the Src Activation loop. (B) Tyrosine phosphorylation of Src as determined by Western blot (total phosphor-Tyr) and mass spectrometry (Y416 on Src A-Loop). FIGURE 5. Carboxyl group footprinting correlates with changes in surface area. Extents of GEE labeling along the (A) HER2 and (B) HER3 dimer interfaces alongside percent changes in solvent accessible surface area (SASA) as determined by MD simulation of HER2-HER3 dimer relative to monomer.


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FIGURE 6. Structural model of HER2-HER3 asymmetric heterodimer subjected to molecular dynamics simulations, highlighting select hydrogen bonds involved in the dimer interface, catalytic loop (C-loop) and stabilization of the activation loop open conformation. FIGURE 7. Effect of HER2 andHER3 kinase domain mutations on specific activity. (A) Specific Activities of HER2 WT and mutants Her2 D769A/H/Y in solution and dimerized with HER3 WT. (B) HER3 WT and mutants HER3 E909G/A/K with fold change calculated relative to HER2 WT alone with no liposomes. (C) HER3 WT and mutants HER3 R948A/K with fold-change calculated relative to HER2 WT alone with no liposomes. Student’s t-test were performed for pairwise comparison of HER3 WT with either R948K or R9489A. TABLE 1. Selected H-bonds from MD Analysis of HER2-HER3 Heterodimer.


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FIGURE 3

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TABLE1

Interaction Potential Function

HER2 K716-HER3 E909 HER2-HER3 Dimer Interface HER2 E717-HER3 K907

HER2 D845-R849 Catalytic Loop Coordination with ATP

HER2 D863-K753 Catalytic Site Coordination with Mg2+ and ATP

Stabilizes HER2 Activation Loop Open Conformation

HER2 E876-R898 HER2 Y877-F899 HER2 E770-L866 HER2 W888-E914 HER2 D871-HER3 R948 HER2 E892-R966

Secures C-terminus of A-Loop

HER2 E892-S903 HER2 E892-R898 HER2 S893-R898


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BoseTimothy S. Collier, Karthikeyan Diraviyam, John Monsey, Wei Shen, David Sept and Ron

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

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http://www.jbc.org/lookup/doi/10.1074/jbc.M113.474882

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http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2013/07/10/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2013/07/10/jbc.M113.474882

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2013/07/10/M113.474882.DC1

http://www.jbc.org/content/suppl/2013/08/29/M113.474882.DCAuthor_profile

http://www.jbc.org/

jbc.m113.474882 key interactions at the her2-her3 dimer interface · 2013-07-10 · key...

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