Allosteric activation of preformed EGF receptor dimers by a single ligand binding event

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane a-helix, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the inactive, symmetric kinase dimer spontaneously activate the receptor in vivo. Optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Allosteric activation of preformed EGF receptor dimers by binding of a single ligand

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Structures of the active HER2/HER3 receptor complex reveal dynamics at the dimerization interface induced by binding of a single ligand

The Human Epidermal Growth Factor Receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex 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. We isolated the NRG1β-bound near full-length HER2/HER3 dimer and obtained a 2.9Å cryo-electron microscopy (cryo-EM) reconstruction of the extracellular domain module which reveals unexpected dynamics at the HER2/HER3 dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved likely because the apo HER2 monomer fails to undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm binding pocket. In a second structure of an oncogenic extracellular domain mutant of HER2, S310F, we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. We show that 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 3.5Å structure of the HER2-S310F/HER3/NRG1β/trastuzumab Fragment antigen binding (Fab) complex shows that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, like oncogenic mutations, therapeutics 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.

A DNA Aptamer That Inhibits the Formation of Unliganded Receptor Dimer and Ligand-Independent Signaling in Cancer Cells

<p>Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, in cancer cells, the overexpression of receptors often causes the formation of unliganded receptor dimers, which can be activated in a ligand-independent manner. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that inhibits ligand-independent receptor activation via preventing the formation of unliganded receptor dimer. By biasing the receptor monomer–dimer equilibrium to the monomer, this aptamer inhibited aberrant cell signaling caused by the unliganded receptor dimer. This work presents a new possibility of oligonucleotide-based therapeutics for cancer.</p>

A DNA Aptamer That Inhibits the Formation of Unliganded Receptor Dimer and Ligand-Independent Signaling in Cancer Cells

<p>Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, in cancer cells, the overexpression of receptors often causes the formation of unliganded receptor dimers, which can be activated in a ligand-independent manner. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that inhibits ligand-independent receptor activation via preventing the formation of unliganded receptor dimer. By biasing the receptor monomer–dimer equilibrium to the monomer, this aptamer inhibited aberrant cell signaling caused by the unliganded receptor dimer. This work presents a new possibility of oligonucleotide-based therapeutics for cancer.</p>

Bacterial Superantigen Toxins, CD28, and Drug Development

During severe bacterial infections, death and disease are often caused by an overly strong immune response of the human host. Acute toxic shock is induced by superantigen toxins, a diverse set of proteins secreted by Gram-positive staphylococcal and streptococcal bacterial strains that overstimulate the inflammatory response by orders of magnitude. The need to protect from superantigen toxins led to our discovery that in addition to the well-known MHC class II and T cell receptors, the principal costimulatory receptor, CD28, and its constitutively expressed coligand, B7-2 (CD86), previously thought to have only costimulatory function, are actually critical superantigen receptors. Binding of the superantigen into the homodimer interfaces of these costimulatory receptors greatly enhances B7-2/CD28 engagement, leading to excessive pro-inflammatory signaling. This finding led to the design of short receptor dimer interface mimetic peptides that block the binding of superantigen and thus protect from death. It then turned out that such a peptide will protect also from Gram-negative bacterial infection and from polymicrobial sepsis. One such CD28 mimetic peptide is advancing in a Phase 3 clinical trial to protect from lethal wound infections by flesh-eating bacteria. These host-oriented therapeutics target the human immune system itself, rendering pathogens less likely to become resistant.

Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation

Cytokines and interferons initiate intracellular signaling via receptor dimerization and activation of Janus kinases (JAKs). How JAKs structurally respond to changes in receptor conformation induced by ligand binding is not known. Here, we present two crystal structures of the human JAK2 FERM and SH2 domains bound to Leptin receptor (LEPR) and Erythropoietin receptor (EPOR), which identify a novel dimeric conformation for JAK2. This 2:2 JAK2/receptor dimer, observed in both structures, identifies a previously uncharacterized receptor interaction essential to dimer formation that is mediated by a membrane-proximal peptide motif called the ‘switch’ region. Mutation of the receptor switch region disrupts STAT phosphorylation but does not affect JAK2 binding, indicating that receptor-mediated formation of the JAK2 FERM dimer is required for kinase activation. These data uncover the structural and molecular basis for how a cytokine-bound active receptor dimer brings together two JAK2 molecules to stimulate JAK2 kinase activity.