HER2/HER3/NRG1b Heterocomplex Structure and Assembly

Cell communication is essential for cellular function and relies on the faithful transmission of signals across the plasma membrane through membrane receptors. Receptor kinases constitute an important class of molecular antennas in which the extracellular signal binding module is linked to an intracellular kinase along one polypeptide chain. Perturbations in this finely coordinated system causes aberrant signaling which lead to pathological states such as cancer or developmental disorders. Despite the disease relevance and extensive therapeutic focus, we fundamentally do not understand how receptor kinases transmit a signal across the plasma membrane in the absence of full-length structures. This is particularly true for the Human Epidermal Growth Factor Receptor 2 (HER2), an orphan receptor, and the Human Epidermal Growth Factor Receptor 3 (HER3), a pseudokinase receptor which form a potent pro-oncogenic heterocomplex upon binding to extracellular ligand. Here, we present three novel high-resolution cryo-electron microscopy (cryo-EM) structures of the extracellular domain of the breast cancer receptor, HER2, engaged with its liganded co-receptor, HER3, solved in the context of near full-length receptors. As the first singly-liganded human HER receptor structures, our findings provide a missing link in the HER receptor field, offer the dimerization arm as an allosteric sensor for ligand binding, visualize HER3 in an extended state for the first time, demonstrate how the most frequent oncogenic HER2 variant, HER2 S310F, exploits dimerization arm dynamics to enhance heterodimerization, and unveil previously unknown details on how commonly prescribed biologic agents bind the heterodimer. Our studies on near full-length HER2 and HER3, when isolated alone, surprisingly reveal that HER2 is a homodimer that may adopt an autoinhibited state and HER3, contrary to dogma, homodimerizes in the presence of NRG1b. Taken together, these findings made possible through the lens of full-length receptor biophysics, explain ligand allostery, inform rational drug design, and add nuance to a model of HER2/HER3/NRG1b heterocomplex assembly

HER2/HER3/NRG1b Heterocomplex Structure and Assembly

Cell communication is essential for cellular function and relies on the faithful transmission of signals across the plasma membrane through membrane receptors. Receptor kinases constitute an important class of molecular antennas in which the extracellular signal binding module is linked to an intracellular kinase along one polypeptide chain. Perturbations in this finely coordinated system causes aberrant signaling which lead to pathological states such as cancer or developmental disorders. Despite the disease relevance and extensive therapeutic focus, we fundamentally do not understand how receptor kinases transmit a signal across the plasma membrane in the absence of full-length structures. This is particularly true for the Human Epidermal Growth Factor Receptor 2 (HER2), an orphan receptor, and the Human Epidermal Growth Factor Receptor 3 (HER3), a pseudokinase receptor which form a potent pro-oncogenic heterocomplex upon binding to extracellular ligand. Here, we present three novel high-resolution cryo-electron microscopy (cryo-EM) structures of the extracellular domain of the breast cancer receptor, HER2, engaged with its liganded co-receptor, HER3, solved in the context of near full-length receptors. As the first singly-liganded human HER receptor structures, our findings provide a missing link in the HER receptor field, offer the dimerization arm as an allosteric sensor for ligand binding, visualize HER3 in an extended state for the first time, demonstrate how the most frequent oncogenic HER2 variant, HER2 S310F, exploits dimerization arm dynamics to enhance heterodimerization, and unveil previously unknown details on how commonly prescribed biologic agents bind the heterodimer. Our studies on near full-length HER2 and HER3, when isolated alone, surprisingly reveal that HER2 is a homodimer that may adopt an autoinhibited state and HER3, contrary to dogma, homodimerizes in the presence of NRG1b. Taken together, these findings made possible through the lens of full-length receptor biophysics, explain ligand allostery, inform rational drug design, and add nuance to a model of HER2/HER3/NRG1b heterocomplex assembly

An effective strategy for ligand-mediated pulldown of the HER2/HER3/NRG1β heterocomplex and cryo-EM structure determination at low sample concentrations.

Obtaining high-resolution structures of Receptor Tyrosine Kinases that visualize extracellular, transmembrane and intracellular kinase regions simultaneously is an eagerly pursued but still unmet challenge of structural biology. The Human Epidermal Growth Factor Receptor 3 (HER3) that has a catalytically inactive kinase domain (pseudokinase) forms a potent signaling complex upon binding of growth factor neuregulin 1β (NRG1β) and upon dimerization with a close homolog, the HER2 receptor. The HER2/HER3/NRG1β complex is often referred to as an oncogenic driver in breast cancer and is an attractive target for anti-cancer therapies. After overcoming significant hurdles in isolating sufficient amounts of the HER2/HER3/NRG1β complex for structural studies by cryo-electron microscopy (cryo-EM), we recently obtained the first high-resolution structures of the extracellular portion of this complex. Here we describe a step-by-step protocol for obtaining a stable and homogenous HER2/HER3/NRG1β complex for structural studies and our recommendation for collecting and processing cryo-EM data for this sample. We also show improved EM density for the transmembrane and kinase domains of the receptors, which continue to evade structural determination at high resolution. The discussed strategies are tunable and applicable to other membrane receptor complexes

Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3.

Biochemical analyses of membrane receptor kinases have been limited by challenges in obtaining sufficient homogeneous receptor samples for downstream structural and biophysical characterization. Here, we report a suite of methods for the efficient expression, purification, and visualization by cryo-electron microscopy (cryo-EM) of near full-length Human Epidermal Growth Factor Receptor 3 (HER3), a receptor tyrosine pseudokinase, in the unliganded state. Through transient mammalian cell expression, a two-step purification with detergent exchange into lauryl maltose neopentyl glycol (LMNG), and freezing devoid of background detergent micelle, we obtained ~6Å reconstructions of the ~60kDa fully-glycosylated unliganded extracellular domain of HER3 from just 30mL of suspension culture. The reconstructions reveal previously unappreciated extracellular domain dynamics and glycosylation sites

Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

Human Epidermal growth factor Receptor 4 (HER4 or ERBB4) carries out essential functions in the development and maintenance of the cardiovascular and nervous systems. HER4 activation is regulated by a diverse group of extracellular ligands including the neuregulin (NRG) family and betacellulin (BTC), which promote HER4 homodimerization or heterodimerization with other HER receptors. Important cardiovascular functions of HER4 are exerted via heterodimerization with its close homolog and orphan receptor, HER2. To date structural insights into ligand-mediated HER4 activation have been limited to crystallographic studies of HER4 ectodomain homodimers in complex with NRG1β. Here, we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1β and BTC. We show that the structures of the heterodimers bound to either ligand are nearly identical and that in both cases the HER2/HER4 heterodimer interface is less dynamic than those observed in structures of HER2/EGFR and HER2/HER3 heterodimers. In contrast, structures of full-length HER4 homodimers bound to NRG1β and BTC display more large-scale dynamics mirroring states previously reported for EGFR homodimers. Our structures also reveal the presence of multiple glycan modifications within HER4 ectodomains, modeled for the first time in HER receptors, that distinctively contribute to the stabilization of HER4 homodimer interfaces over those of HER2/HER4 heterodimers

Structures of the HER2-HER3-NRG1β complex reveal a dynamic dimer interface.

Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex1-3 upon binding of growth factor neuregulin-1β (NRG1β). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. 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. 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. 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. 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. Our structure of the HER2(S310F)-HER3-NRG1β-trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2-HER3 heterodimer. 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

Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3

Abstract PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity

Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3.

PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity