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Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs., Bone morphogenetic protein (BMP) receptors are single pass transmembrane serine/threonine kinases that form tetrameric complexes comprised of two type I and two type II BMP receptors. Here the authors characterize a structure of an active type I/type II kinase tetramer providing insight into molecular mechanism driving ligand-induced signaling.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

The protein JAK2 is a prototypical member of the Janus kinase family, and mediates signals from numerous cytokine receptors. The constitutively active V617F mutant of JAK2 is prevalent in many bone marrow disorders, blood cancers, and autoimmune diseases, and is an important drug target. Structures have been determined for each of the four individual domains making up JAK2, and for certain pairs of these domains, but no structure of full-length JAK2 is available, and thus the mechanisms underlying JAK2 regulation and the aberrant activity of the V617F mutant have been incompletely understood. Here we propose structural models of full-length JAK2 in both its active and inactive forms. Construction of these models was informed by long-timescale molecular dynamics simulations. Subsequent mutagenesis experiments showed that mutations at the putative interdomain interfaces modulated JAK2 activity. The models provide a structural basis for understanding JAK2 autoinhibition and activation, and suggest that the constitutive activity of the V617F mutant may arise from a dual effect of destabilizing the inactive conformation and stabilizing the active conformation.

Trans-autophosphorylation is among the most prevalent means of protein kinase activation, yet its molecular basis is poorly defined. In Toll-like receptor and interleukin-1 receptor signaling pathways, the kinase IRAK4 is recruited to the membrane-proximal adaptor MyD88 through death domain (DD) interactions, forming the oligomeric Myddosome and mediating NF-κB activation. Here we show that unphosphorylated IRAK4 dimerizes in solution with a KD of 2.5 μM and that Myddosome assembly greatly enhances IRAK4 kinase domain (KD) autophosphorylation at sub-KD concentrations. The crystal structure of the unphosphorylated IRAK4(KD) dimer captures a conformation that appears to represent the actual trans-autophosphorylation reaction, with the activation loop phosphosite of one IRAK4 monomer precisely positioned for phosphotransfer by its partner. We show that dimerization is crucial for IRAK4 autophosphorylation in vitro and ligand-dependent signaling in cells. These studies identify a mechanism for oligomerization-driven allosteric autoactivation of IRAK4 that may be general to other kinases activated by autophosphorylation.

SummaryHow the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7-kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity., Protein tyrosine kinases are subject to multiple regulatory mechanisms. Foda et al. show that reactants and products of the tyrosine kinase Src bind its catalytic domain with opposite cooperativity, and identify an allosteric network of dynamically coupled amino acids that underlie this behaviour.

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001, eLife digest Hormones are chemicals that are produced to carry signals around the body. Mammals, including humans, need hormones called insulin and insulin-like growth factors (or IGF for short) to grow and develop normally. These hormones bind to, and activate, specific proteins—known as receptors—that span from the outside of the cell to the inside through the cell's surface membrane. The insulin and IGF receptors are complex molecules, each composed of two identical protein subunits that are linked together. The hormones bind to the part of the proteins (known as the extracellular domains) that are on the outside surface of cells. When no hormone is bound to the receptor, these two extracellular domains form an inverted ‘V’ shape and the two regions that cross the membrane are held far apart. It was unclear, however, how this shape changes when the hormone binds, and how this change in shape activates the receptor. Kavran et al. have now compared the known three-dimensional structures of the extracellular domains of the insulin receptor, both with and without a molecule of insulin bound to it. This comparison highlighted an interaction between the two receptor subunits that was disrupted when insulin was bound to the receptor. This interaction appeared to stabilize the inverted ‘V’ structure and hold apart the parts of the proteins that span the surface—which in turn separates the regions of the proteins that are inside the cell. Kavran et al. suggest that this separation keeps the insulin receptor in an inactive state. Removing either the whole extracellular domain of the IGF receptor—or a smaller portion that keeps the regions that cross the cell membrane separate—resulted in the receptor being activated, even when insulin-like growth factor was not bound to the receptor. Kavran et al. then confirmed that when a molecule of insulin-like growth factor binds to the extracellular domain of the IGF receptor, it causes a shape change that moves the parts of the receptor that span the cell membrane closer together. This results in the regions of the receptor proteins located inside the cell adding chemical tags, called phosphate groups, to one another—which activates the receptor. When there are problems with how these receptors are activated or inactivated in humans, serious disorders including diabetes and cancer can occur. As such, the findings of Kavran et al. might help future work aimed at regulating the activation of the insulin and IGF receptors to treat these and other diseases. DOI: http://dx.doi.org/10.7554/eLife.03772.002

Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using unbiased molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivity of V617F, the predominant JAK2 MPN mutation.

The human epidermal growth factor receptor (HER) tyrosine kinases homo- and heterodimerize to activate downstream signaling pathways. HER3 is a catalytically impaired member of the HER family that contributes to the development of several human malignancies and is mutated in a subset of cancers. HER3 signaling depends on heterodimerization with a catalytically active partner, in particular epidermal growth factor receptor (EGFR) (the founding family member, also known as HER1) or HER2. The activity of homodimeric complexes of catalytically active HER family members depends on allosteric activation between the two kinase domains. To determine the structural basis for HER3 signaling through heterodimerization with a catalytically active HER family member, we solved the crystal structure of the heterodimeric complex formed by the isolated kinase domains of EGFR and HER3. The structure showed HER3 as an allosteric activator of EGFR and revealed a conserved role of the allosteric mechanism in activation of HER family members through heterodimerization. To understand the effects of cancer-associated HER3 mutations at the molecular level, we solved the structures of two kinase domains of HER3 mutants, each in a heterodimeric complex with the kinase domain of EGFR. These structures, combined with biochemical analysis and molecular dynamics simulations, indicated that the cancer-associated HER3 mutations enhanced the allosteric activator function of HER3 by redesigning local interactions at the dimerization interface.

The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo., Author Summary Epidermal growth factor receptor (EGFR) molecules are of central importance in cellular communication. Embedded in the cell membrane, these receptors bind epidermal growth factor (EGF) molecules outside the cell and translate this binding into specific biochemical signals inside the cell, which in turn trigger cell proliferation, migration, or differentiation. EGFR dysfunction has been implicated in a variety of cancers, and EGFR-targeting drugs are commonly used in cancer treatments. It has been widely assumed that the extracellular portion of an EGFR molecule protrudes perpendicularly from the cell membrane. In detailed, atomic-level computer simulations, however, we find that it lies down on the membrane, placing its EGF-binding site adjacent to the membrane surface. We further show that EGF may interact with EGFR in two distinct ways (with or without the involvement of the membrane). This may explain the experimental finding that an EGF molecule binds to EGFR more weakly at higher EGF concentration. This phenomenon, which is a manifestation of an underlying negative cooperativity, is an important but poorly understood characteristic of EGFR activity. In this study, we also model and analyze the glycan chains attached to EGFR, which are integral to its behavior in living cells.

The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human. DOI: http://dx.doi.org/10.7554/eLife.00708.001, eLife digest ErbB proteins are found in most multi-cellular organisms, and are involved in the regulation of a number of important cellular processes, including proliferation, migration, and differentiation. Humans have four ErbB proteins, which span the plasma membrane of cells. These proteins respond to interactions with molecules outside the cell—such as growth factors and hormones—by sending signals along the appropriate signaling pathway within the cell. ErbB proteins have three portions: an ectodomain that extends outside the cell; a single helix that spans the membrane; and a cytoplasmic domain inside the cell. When a signaling ligand molecule outside the cell binds to the ectodomain of an ErbB protein, this protein must then combine with another ErbB protein to form a dimer before a signal can be sent within the cell. These dimers can include two copies of the same ErbB protein or two different ErbB proteins. However, one of the ErbB proteins—Her2—works in a different way. It cannot bind ligands outside the cell, and it can only send a signal within the cell if it first forms a dimer with an ErbB protein of another type, which itself must be bound to an external ligand. The four ErbB proteins diverged from a common ancestor relatively recently, yet they are now diverse enough to play key roles in a variety of complex signaling networks. In particular, the fact that Her2 cannot bind external ligands, and that it must form a dimer with a different ErbB protein before it can send a signal, has led to suggestions that the role of Her2 is to amplify the signals from other ErbB proteins. Since high levels of Her2 are associated with aggressive forms of breast and ovarian cancer, understanding how it is activated could improve our understanding of these cancers. Arkhipov et al. have now used computer simulations to model how Her2 forms dimers with other ErbB proteins in human cells. They based these simulations on crystal structures of human ErbB proteins and dEGFR, a growth-factor receptor found in fruit flies that closely resembles the ErbB proteins found in humans. They found that the dimers were stable as long as one protein within the dimer was bound to a ligand. Removing this ligand, however, distorted the ectodomain of the host protein, creating a gap that weakened the dimer and prevented Her2 from sending a signal within the cell. Similar results were obtained with the fruit fly dEGFR proteins. These simulations suggest that ErbB proteins form dimers and send signals through a mechanism conserved in evolution. Research in this field might help ongoing efforts to develop new treatments for human tumors characterized by high levels of Her2 expression. DOI: http://dx.doi.org/10.7554/eLife.00708.002