RESUMO
ABSTRACT: Secondary kinase domain mutations in BCR::ABL1 represent the most common cause of resistance to tyrosine kinase inhibitor (TKI) therapy in patients with chronic myeloid leukemia. The first 5 approved BCR::ABL1 TKIs target the adenosine triphosphate (ATP)-binding pocket. Mutations confer resistance to these ATP-competitive TKIs and those approved for other malignancies by decreasing TKI affinity and/or increasing ATP affinity. Asciminib, the first highly active allosteric TKI approved for any malignancy, targets an allosteric regulatory pocket in the BCR::ABL1 kinase C-lobe. As a non-ATP-competitive inhibitor, the activity of asciminib is predicted to be impervious to increases in ATP affinity. Here, we report several known mutations that confer resistance to ATP-competitive TKIs in the BCR::ABL1 kinase N-lobe that are distant from the asciminib binding pocket yet unexpectedly confer in vitro resistance to asciminib. Among these is BCR::ABL1 M244V, which confers clinical resistance even to escalated asciminib doses. We demonstrate that BCR::ABL1 M244V does not impair asciminib binding, thereby invoking a novel mechanism of resistance. Molecular dynamic simulations of the M244V substitution implicate stabilization of an active kinase conformation through impact on the α-C helix as a mechanism of resistance. These N-lobe mutations may compromise the clinical activity of ongoing combination studies of asciminib with ATP-competitive TKIs.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Inibidores de Proteínas Quinases , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Trifosfato de Adenosina/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-abl/química , Niacinamida/análogos & derivados , PirazóisRESUMO
Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.
Assuntos
Antineoplásicos , Piperazinas , Mesilato de Imatinib , Piperazinas/farmacologia , Pirimidinas/farmacologia , Benzamidas , Antineoplásicos/farmacologia , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-ablRESUMO
Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often "cryptic" without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein-ligand systems.
Assuntos
Trifosfato de Adenosina , Simulação por Computador , Inibidores de Proteínas Quinases , Quinases da Família src , Trifosfato de Adenosina/química , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Humanos , Ligantes , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/químicaRESUMO
Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are "cryptic": When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)-which performs its function by forming a PPI with its receptor-without incorporating any prior structural information about the ligands' binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein-small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2-small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.
Assuntos
Descoberta de Drogas , Interleucina-2 , Sítios de Ligação , Interleucina-2/química , Interleucina-2/metabolismo , Ligantes , Ligação ProteicaRESUMO
Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques.
Assuntos
Neoplasias da Mama/metabolismo , Carcinogênese/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Compostos de Anilina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Nitrilas/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Galectinas/química , Galectinas/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese , Multimerização Proteica , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Receptores de Ativinas Tipo I/química , Receptores de Ativinas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Transdução de Sinais/fisiologia , Receptores de Ativinas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Humanos , Ligantes , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Domínios Proteicos , Hipertensão Arterial Pulmonar , Espalhamento a Baixo Ângulo , Transdução de Sinais/genética , Proteínas Smad/metabolismo , Difração de Raios XRESUMO
Plexins are semaphorin receptors that play essential roles in mammalian neuronal axon guidance and in many other important mammalian biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism and is modulated by small GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations, we showed that RND1 reinforces the plexin dimerization interface, whereas RhoD destabilizes it due to their differential interaction with the cell membrane. Upon binding plexin at the Rho-GTPase-binding domain (RBD), RND1 and RhoD interact differently with the inner leaflet of the cell membrane and exert opposite effects on the dimerization interface via an allosteric network involving the RBD, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively charged membrane interface.
Assuntos
Semaforinas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Axônios/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Neurônios/metabolismo , Ligação Proteica , Multimerização Proteica , Transdução de Sinais , Proteínas rho de Ligação ao GTP/químicaRESUMO
AZD9291 (Osimertinib) is highly effective in treating EGFR-mutated non-small-cell lung cancers (NSCLCs) with T790M-mediated drug resistance. Despite the remarkable success of AZD9291, its binding pose with EGFR T790M remains unclear. Here, we report unbiased, atomic-level molecular dynamics (MD) simulations in which spontaneous association of AZD9291 with EGFR kinases having WT and T790M mutant gatekeepers was observed. Simulation-generated structural models suggest that the binding pose of AZD9291 with T790M differs from its binding pose with the WT, and that AZD9291 interacts extensively with the gatekeeper residue (Met 790) in T790M but not with Thr 790 in the WT, which explains why AZD9291 binds T790M with higher affinity. The MD simulation-generated models were confirmed by experimentally determined EGFR/T790M complex crystal structures. This work may facilitate the rational design of drugs that can overcome resistance mutations to AZD9291, and more generally it suggests the potential of using unbiased MD simulation to elucidate small-molecule binding poses.
Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/química , Compostos de Anilina/química , Cristalografia por Raios X , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/químicaRESUMO
Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Multimerização Proteica , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Matriz Extracelular/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ligantes , Modelos Biológicos , Modelos Moleculares , Fotodegradação , Polímeros/química , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/metabolismoRESUMO
Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.
Assuntos
Receptores ErbB/química , Receptores ErbB/metabolismo , Animais , Artefatos , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Fator de Crescimento Epidérmico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ligantes , Simulação de Dinâmica Molecular , Fosforilação , Domínios Proteicos , Multimerização Proteica , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Toxoplasma/enzimologia , Regulação Alostérica , Animais , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/metabolismo , Anticorpos Antiprotozoários/farmacologia , Biocatálise/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Camelídeos Americanos , Células Cultivadas , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Toxoplasma/genéticaRESUMO
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.
Assuntos
Quinases da Família src/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/química , Quinases da Família src/genéticaRESUMO
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.
Assuntos
Receptores ErbB/metabolismo , Modelos Moleculares , Mutação/genética , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Transdução de Sinais/fisiologia , Clonagem Molecular , Cristalização , Dimerização , Receptores ErbB/química , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Receptor ErbB-3/química , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/química , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , Domínio Catalítico , Células Cultivadas , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Espalhamento de Radiação , Especificidade por SubstratoRESUMO
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.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Receptor de Insulina/química , Receptor de Insulina/metabolismoRESUMO
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.
Assuntos
Membrana Celular/química , Receptores ErbB/química , Receptores ErbB/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Animais , Sítios de Ligação , Galinhas , Biologia Computacional , Ligantes , Mamíferos , Simulação de Dinâmica Molecular , Conformação Proteica , Peixe-ZebraRESUMO
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 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.
Assuntos
Janus Quinase 2/fisiologia , Modelos Genéticos , Sítios de Ligação , Simulação por Computador , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Modelos Moleculares , Estrutura Terciária de Proteína , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Structurally elucidating transition pathways between protein conformations gives deep mechanistic insight into protein behavior but is typically difficult. Unbiased molecular dynamics (MD) simulations provide one solution, but their computational expense is often prohibitive, motivating the development of enhanced sampling methods that accelerate conformational changes in a given direction, embodied in a collective variable. The accuracy of such methods is unclear for complex protein transitions, because obtaining unbiased MD data for comparison is difficult. Here, we use long-time scale, unbiased MD simulations of epidermal growth factor receptor kinase deactivation as a complex biological test case for two widely used methods-steered molecular dynamics (SMD) and the string method. We found that common collective variable choices, based on the root-mean-square deviation (RMSD) of the entire protein, prevented the methods from producing accurate paths, even in SMD simulations on the time scale of the unbiased transition. Using collective variables based on the RMSD of the region of the protein known to be important for the conformational change, however, enabled both methods to provide a more accurate description of the pathway in a fraction of the simulation time required to observe the unbiased transition.
RESUMO
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.