Rod-shaped density consistent with the asymmetric kinase domain dimer is visible below the micelle. f, Near full-length reconstruction of HER2-S310F/HER3/NRG1β after particle recentering with center of mass at the micelle in RELION from a ~45,000 particle stack. e, Representative negative stain electron microscopy 2D class averages of sample from HER2/HER3/NRG1β complex fractions (ECDs (extracellular domains), ICDs (intracellular domains)).
The mass is consistent with that of the HER2/HER3/NRG1β complex (predicted ~280 kDa without accounting for micelle mass, ~340 kDa assuming a ~60 kDa DDM micelle mass).
d, Mass photometry of the peak sample indicates that the majority of particles have an average mass of ~326 kDa with a standard deviation of 17 kDa. We routinely obtained a 1–2 mAu peaks for the complex preparations from 120 ml of mammalian culture, which was sufficient for the structural studies. The major peak at ~14 ml elution volume is marked grey and corresponds to the fractions used for structural studies. c, Representative size exclusion chromatography profile of the HER2/HER3/NRG1β complex resolved on a Superose6 10/300 Increase column (GE Healthcare). The gel is representative of three independent experiments. b, A representative Coomassie-stained SDS-PAGE gel analysis of the HER2/HER3/NRG1β complex after purification showing bands corresponding to the HER2 and HER3 receptors. The Author(s), under exclusive licence to Springer Nature Limited.Ī, Schematic summary of the HER2/HER3/NRG1β complex purification strategy. The unique features of a singly liganded HER2-HER3 heterodimer underscore the allosteric sensing of ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via a canonical active dimer interface. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2-HER3 heterodimer. Our structure of the HER2(S310F)-HER3-NRG1β-trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Both HER2-HER3 and HER2(S310F)-HER3 retain the capacity to bind to the HER2-directed therapeutic antibody trastuzumab, but the mutant complex does not bind to pertuzumab. In a structure of the oncogenic extracellular domain mutant HER2(S310F), we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved because the apo HER2 monomer does not undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm-binding pocket. Here we isolated the NRG1β-bound near full-length HER2-HER3 dimer and, using cryo-electron microscopy, reconstructed the extracellulardomain module, revealing unexpected dynamics at the HER2-HER3 dimerization interface. The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex 1-3 upon binding of growth factor neuregulin-1β (NRG1β).
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