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 NRG1ß. Here, we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1ß 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 NRG1ß 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.

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.

An effective strategy for ligand-mediated pulldown of the HER2/HER3/NRG1ß 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 1ß (NRG1ß) and upon dimerization with a close homolog, the HER2 receptor. The HER2/HER3/NRG1ß 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/NRG1ß 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/NRG1ß 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.

Structures of the HER2-HER3-NRG1ß 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-1ß (NRG1ß). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Here we isolated the NRG1ß-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 NRG1ß-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-NRG1ß-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.

Drugging the "Undruggable" MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.

How Hsp90 and Cdc37 Lubricate Kinase Molecular Switches.

The Hsp90/Cdc37 chaperone system interacts with and supports 60% of the human kinome. Not only are Hsp90 and Cdc37 generally required for initial folding, but many kinases rely on the Hsp90/Cdc37 throughout their lifetimes. A large fraction of these 'client' kinases are key oncoproteins, and their interactions with the Hsp90/Cdc37 machinery are crucial for both their normal and malignant activity. Recently, advances in single-particle cryo-electron microscopy (cryoEM) and biochemical strategies have provided the first key molecular insights into kinase-chaperone interactions. The surprising results suggest a re-evaluation of the role of chaperones in the kinase lifecycle, and suggest that such interactions potentially allow kinases to more rapidly respond to key signals while simultaneously protecting unstable kinases from degradation and suppressing unwanted basal activity.

Cross-monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity.

The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown. Here, we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drives an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity.