RESUMEN
The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.
Asunto(s)
Receptores ErbB/química , Receptores ErbB/genética , Neoplasias/metabolismo , Mutación Puntual , Transducción de Señal , Secuencia de Aminoácidos , Cristalografía por Rayos X , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Gefitinib , Humanos , Lapatinib , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Pliegue de Proteína , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Estructura Terciaria de Proteína , Quinazolinas/farmacología , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Descubrimiento de Drogas , Interleucina-2 , Sitios de Unión , Interleucina-2/química , Interleucina-2/metabolismo , Ligandos , Unión ProteicaRESUMEN
The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the so-called "Src-like inactive" conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed "extended" conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another ["Asp-Phe-Gly-out" ("DFG-out")] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or "cracking," which supports a prediction of energy landscape theory. We used hydrogen-deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.
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Biocatálisis , Receptores ErbB/química , Secuencias de Aminoácidos , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Activación Enzimática , Receptores ErbB/metabolismo , Simulación de Dinámica Molecular , Estructura Secundaria de Proteína , Familia-src Quinasas/química , Familia-src Quinasas/metabolismoRESUMEN
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.
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Membrana Celular/química , Receptores ErbB/química , Receptores ErbB/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Animales , Sitios de Unión , Pollos , Biología Computacional , Ligandos , Mamíferos , Simulación de Dinámica Molecular , Conformación Proteica , Pez CebraRESUMEN
Members of the RAS small GTPase family regulate cellular responses to extracellular stimuli by mediating the flux through downstream signal transduction cascades. RAS activity is strongly dependent on its subcellular localization and its nucleotide-binding status, both of which are modulated by posttranslational modification. We have determined that RAS is posttranslationally acetylated on lysine 104. Molecular dynamics simulations suggested that this modification affects the conformational stability of the Switch II domain, which is critical for the ability of RAS to interact with guanine nucleotide exchange factors. Consistent with this model, an acetylation-mimetic mutation in K-RAS4B suppressed guanine nucleotide exchange factor-induced nucleotide exchange and inhibited in vitro transforming activity. These data suggest that lysine acetylation is a negative regulatory modification on RAS. Because mutations in RAS family members are extremely common in cancer, modulation of RAS acetylation may constitute a therapeutic approach.
Asunto(s)
Genes ras/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas ras/química , Proteínas ras/metabolismo , Acetilación , Animales , Células COS , Chlorocebus aethiops , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Prenilación/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/fisiología , Relación Estructura-Actividad , Proteínas ras/genéticaRESUMEN
INTRODUCTION: Surgical counseling enables shared decision making and optimal outcomes by improving patients' understanding about their pathologies, surgical options, and expected outcomes. Here, we aimed to provide practical answers to frequently asked questions (FAQs) from patients undergoing an anterior cervical diskectomy and fusion (ACDF) or cervical disk replacement (CDR) for the treatment of degenerative conditions. METHODS: Patients who underwent primary one-level or two-level ACDF or CDR for the treatment of degenerative conditions with a minimum of 1-year follow-up were included. Data were used to answer 10 FAQs that were generated from author's experience of commonly asked questions in clinic before ACDF or CDR. RESULTS: A total of 395 patients (181 ACDF, 214 CDR) were included. (1, 2, and 3) Will my neck/arm pain and physical function improve? Patients report notable improvement in all patient-reported outcome measures. (4) Is there a chance I will get worse? 13% (ACDF) and 5% (CDR) reported worsening. (5) Will I receive a significant amount of radiation? Patients on average received a 3.7 (ACDF) and 5.5 mGy (CDR) dose during. (6) How long will I stay in the hospital? Most patients get discharged on postoperative day one. (7) What is the likelihood that I will have a complication? 13% (8% minor and 5% major) experienced in-hospital complications (ACDF) and 5% (all minor) did (CDR). (8) Will I need another surgery? 2.2% (ACDF) and 2.3% (CDR) of patients required a revision surgery. (9 & 10) When will I be able to return to work/driving? Most patients return to working (median of 16 [ACDF] and 14 days [CDR]) and driving (median of 16 [ACDF] and 12 days [CDR]). CONCLUSIONS: The answers to the FAQs can assist surgeons in evidence-based patient counseling.
RESUMEN
Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, detailed experimental characterization of the process by which such binding occurs has proven challenging. We conducted relatively long, unguided molecular dynamics simulations in which a ligand (the cancer drug dasatinib or the kinase inhibitor PP1) was initially placed at a random location within a box that also contained a protein (Src kinase) to which that ligand was known to bind. In several of these simulations, the ligand correctly identified its target binding site, forming a complex virtually identical to the crystallographically determined bound structure. The simulated trajectories provide a continuous, atomic-level view of the entire binding process, revealing persistent and noteworthy intermediate conformations and shedding light on the role of water molecules. The technique we employed, which does not assume any prior knowledge of the binding site's location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.
Asunto(s)
Simulación de Dinámica Molecular , Pirazoles/metabolismo , Pirimidinas/metabolismo , Tiazoles/metabolismo , Sitios de Unión , Dasatinib , Unión Proteica , Conformación Proteica , Pirazoles/farmacología , Pirimidinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/química , Familia-src Quinasas/metabolismoRESUMEN
The RosettaAntibody server (http://antibody.graylab.jhu.edu) predicts the structure of an antibody variable region given the amino-acid sequences of the respective light and heavy chains. In an initial stage, the server identifies and displays the most sequence homologous template structures for the light and heavy framework regions and each of the complementarity determining region (CDR) loops. Subsequently, the most homologous templates are assembled into a side-chain optimized crude model, and the server returns a picture and coordinate file. For users requesting a high-resolution model, the server executes the full RosettaAntibody protocol which additionally models the hyper-variable CDR H3 loop. The high-resolution protocol also relieves steric clashes by optimizing the CDR backbone torsion angles and by simultaneously perturbing the relative orientation of the light and heavy chains. RosettaAntibody generates 2000 independent structures, and the server returns pictures, coordinate files, and detailed scoring information for the 10 top-scoring models. The 10 models enable users to use rational judgment in choosing the best model or to use the set as an ensemble for further studies such as docking. The high-resolution models generated by RosettaAntibody have been used for the successful prediction of antibody-antigen complex structures.
Asunto(s)
Región Variable de Inmunoglobulina/química , Homología de Secuencia de Aminoácido , Programas Informáticos , Algoritmos , Regiones Determinantes de Complementariedad/química , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Modelos Moleculares , Interfaz Usuario-ComputadorRESUMEN
By means of a combination of genome-wide and follow-up studies, recent large-scale association studies of populations of European descent have now identified over 46 loci associated with coronary artery disease (CAD). As part of the TAICHI Consortium, we have collected and genotyped 8556 subjects from Taiwan, comprising 5423 controls and 3133 cases with coronary artery disease, for 9087 CAD SNPs using the CardioMetaboChip. We applied penalized logistic regression to ascertain the top SNPs that contribute together to CAD susceptibility in Taiwan. We observed that the 9p21 locus contributes to CAD at the level of genome-wide significance (rs1537372, with the presence of C, the major allele, the effect estimate is -0.216, standard error 0.033, p value 5.8x10-10). In contrast to a previous report, we propose that the 9p21 locus is a single genetic contribution to CAD in Taiwan because: 1) the penalized logistic regression and the follow-up conditional analysis suggested that rs1537372 accounts for all of the CAD association in 9p21, and 2) the high linkage disequilibrium observed for all associated SNPs in 9p21. We also observed evidence for the following loci at a false discovery rate >5%: SH2B3, ADAMTS7, PHACTR1, GGCX, HTRA1, COL4A1, and LARP6-LRRC49. We also took advantage of the fact that penalized methods are an efficient approach to search for gene-by-gene interactions, and observed that two-way interactions between the PHACTR1 and ADAMTS7 loci and between the SH2B3 and COL4A1 loci contribute to CAD risk. Both the similarities and differences between the significance of these loci when compared with significance of loci in studies of populations of European descent underscore the fact that further genetic association of studies in additional populations will provide clues to identify the genetic architecture of CAD across all populations worldwide.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple , TaiwánRESUMEN
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.
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Receptores ErbB/química , Receptores ErbB/metabolismo , Animales , Artefactos , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Factor de Crecimiento Epidérmico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ligandos , Simulación de Dinámica Molecular , Fosforilación , Dominios Proteicos , Multimerización de Proteína , Transducción de SeñalRESUMEN
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.
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Familia-src Quinasas/metabolismo , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
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.
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Janus Quinasa 2/fisiología , Modelos Genéticos , Sitios de Unión , Simulación por Computador , Humanos , Janus Quinasa 2/química , Janus Quinasa 2/genética , Modelos Moleculares , Estructura Terciaria de Proteína , Transducción de Señal , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Receptores ErbB/metabolismo , Receptor ErbB-2/metabolismo , Secuencia de Aminoácidos , Dimerización , Receptores ErbB/química , Humanos , Datos de Secuencia Molecular , Receptor ErbB-2/química , Homología de Secuencia de AminoácidoRESUMEN
A molecular description of protein-surface interactions could open new avenues in bionanotechnology and provide a deeper understanding of in vivo phase boundary biophysics. However, current experimental techniques can provide only inferential or incomplete information about the protein-surface interface. We present a novel computational method for modeling the interactions of proteins with solid surfaces using comprehensive sampling and an atomistic description. The approach relies on an all-atom Monte Carlo plus-minimization search algorithm that rapidly and simultaneously optimizes rigid-body and side-chain conformations. We apply the method to the statherin-hydroxyapatite system, an evolved protein-surface interaction that is likely to have one or a few specific structural solutions. The algorithm converges on a set of low energy, entropically favorable structures that are consistent with previous experimental results, namely protein-surface intermolecular distances acquired by solid-state NMR. The simulations isolate particular residues as being primary contributors to the adsorption free energy (hydrogen bonding, van der Waals, and electrostatic energies), in agreement with previous mutagenesis, deletion, and single amino acid experiments. We also report the discovery of a molecular recognition motif where the N-terminal alpha-helix of statherin places all four of its basic residues to match the periodicity of open phosphate triad clusters across the [001] monoclinic face of the hydroxyapatite surface. Results suggest new experiments that could further elucidate the structural features of this important biological system.