RESUMEN
Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.
Asunto(s)
Fosforilación , Utrofina , Animales , Distrofina/genética , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Utrofina/química , Utrofina/genética , Bacterias , Insectos , RatonesRESUMEN
Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.
Asunto(s)
Actinas , Distrofina , Distrofia Muscular de Duchenne , Humanos , Actinas/metabolismo , Distrofina/genética , Distrofina/química , Terapia Genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Dominios ProteicosRESUMEN
Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).
Asunto(s)
Regulación Alostérica/fisiología , Quinasa 2 Dependiente de la Ciclina/metabolismo , Sitio Alostérico/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.
Asunto(s)
Bencilaminas/química , Transferencia Resonante de Energía de Fluorescencia , Miosinas/química , Uracilo/análogos & derivados , Secuencias de Aminoácidos , Animales , Bencilaminas/uso terapéutico , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Cardiomiopatía Hipertrófica/metabolismo , Bovinos , Cinética , Miosinas/metabolismo , Uracilo/química , Uracilo/uso terapéuticoRESUMEN
We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.
Asunto(s)
Actinas/metabolismo , Bencilaminas/farmacología , Miosinas Cardíacas/metabolismo , Cardiomiopatía Hipertrófica Familiar/tratamiento farmacológico , Subfragmentos de Miosina/metabolismo , Uracilo/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Bencilaminas/uso terapéutico , Miosinas Cardíacas/química , Cardiomiopatía Hipertrófica Familiar/etiología , Humanos , Cinética , Subfragmentos de Miosina/química , Estabilidad Proteica/efectos de los fármacos , Uracilo/farmacología , Uracilo/uso terapéuticoRESUMEN
Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure-activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.
Asunto(s)
Aurora Quinasa A/efectos de los fármacos , Aurora Quinasa A/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Regulación Alostérica , Secuencias de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , División Celular , Cristalografía por Rayos X , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Nucleares/metabolismo , Oligopéptidos , Fosforilación , Unión ProteicaRESUMEN
Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these-acetylation of lysine 146 in Eg5-by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site-neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant-features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a "brake" that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.
Asunto(s)
Cinesinas/química , Cinesinas/metabolismo , Mitosis/fisiología , Secuencia de Aminoácidos , Fenómenos Biomecánicos , Células HeLa , Humanos , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ß-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.
Asunto(s)
Mecanotransducción Celular , Actividad Motora , Mutación , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Modelos Moleculares , Miosina Tipo V/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , Homología de SecuenciaRESUMEN
Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.
Asunto(s)
Miosinas Cardíacas/efectos de los fármacos , Insuficiencia Cardíaca/fisiopatología , Miosinas/efectos de los fármacos , Urea/análogos & derivados , Animales , Técnicas Biosensibles , Miosinas Cardíacas/química , Miosinas Cardíacas/aislamiento & purificación , Fármacos Cardiovasculares/administración & dosificación , Fármacos Cardiovasculares/química , Bovinos , Pollos , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Cinética , Contracción Miocárdica/efectos de los fármacos , Miocardio/enzimología , Miocardio/patología , Miosinas/química , Fosfatos/química , Fosfatos/metabolismo , Conejos , Urea/administración & dosificación , Urea/químicaRESUMEN
A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)(2)FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.
Asunto(s)
Proteínas Aviares/química , Modelos Químicos , Miosinas/química , Regulación Alostérica , Animales , Proteínas Aviares/metabolismo , Pollos , Transferencia Resonante de Energía de Fluorescencia , Cinética , Miosinas/metabolismo , ConejosRESUMEN
Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction--including switch-1 and the neck linker--how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as "structural kinetics," to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.
Asunto(s)
Cinesinas/fisiología , Modelos Moleculares , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinesinas/química , Cinética , Microtúbulos/química , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Ovinos , TemperaturaRESUMEN
Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor-acceptor pairs to examine the kinetics of lever arm swing during the recovery and power stroke phases of the ATPase cycle. During the recovery stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the power stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-Å change in distance between the pre- and postpower stroke states and shows that the lever arm is more dynamic in the postpower stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid power stroke that gates the release of phosphate, whereas a second slower power stroke may be important for mediating strain sensitivity.
Asunto(s)
Miosina Tipo V/química , Adenosina Difosfato/química , Adenosina Trifosfato/química , Catálisis , Dominio Catalítico , Transferencia Resonante de Energía de FluorescenciaRESUMEN
We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.
Asunto(s)
Actinas/metabolismo , Dominio Catalítico , Sistemas de Computación , Dictyostelium/metabolismo , Miosina Tipo II/química , Animales , Fenómenos Biomecánicos , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Transferencia Resonante de Energía de Fluorescencia , Cinética , Miosina Tipo II/metabolismo , Fosfatos/metabolismo , Estructura Secundaria de Proteína , Conejos , Factores de TiempoRESUMEN
Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cinesinas/química , Microtúbulos/ultraestructura , Mitosis/genética , Adenosina Trifosfatasas/química , Adenilil Imidodifosfato/química , Sitios de Unión , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinesinas/genética , Cinesinas/ultraestructura , Cinética , Microscopía Electrónica , Microtúbulos/química , Unión Proteica/genética , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock absorber that limits myofiber membrane damage when undergoing reversible unfolding upon muscle stretching and contraction. Utrophin is a dystrophin homologue that is under investigation as a protein replacement therapy for DMD. However, it remains uncertain whether utrophin can mechanically substitute for dystrophin. Here, we compared the mechanical properties of homologous utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using two operational modes of atomic force microscopy (AFM), constant speed and constant force. Our comprehensive data, including the statistics of force magnitude at which the folded domains unfold in constant speed mode and the time of unfolding statistics in constant force mode, show consistent results. We recover parameters of the energy landscape of the domains and conducted Monte Carlo simulations which corroborate the conclusions drawn from experimental data. Our results confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness compared to insect UtrN-R3, while the mechanical stiffness of the homologous region of dystrophin (DysN-R3) is intermediate between bacterial and insect UtrN-R3, showing greater similarity to bacterial UtrN-R3.
RESUMEN
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
Asunto(s)
Inhibidores de Proteínas Quinasas , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Regulación Alostérica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Humanos , Conformación Proteica , Unión Proteica , Modelos MolecularesRESUMEN
We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.
Asunto(s)
Magnesio/química , Miosina Tipo V/química , Nucleótidos de Adenina/química , ADN Complementario , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Miosina Tipo V/genética , Conformación Proteica , TermodinámicaRESUMEN
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
RESUMEN
Fatty acid binding protein 4 (FABP4) is a secreted adipokine linked to obesity and progression of a variety of cancers. Obesity increases extracellular FABP4 (eFABP4) levels in animal models and in obese breast cancer patients compared with lean healthy controls. Using MCF-7 and T47D breast cancer epithelial cells, we show herein that eFABP4 stimulates cellular proliferation in a time and concentration dependent manner while the non-fatty acid-binding mutant, R126Q, failed to potentiate growth. When E0771 murine breast cancer cells were injected into mice, FABP4 null animals exhibited delayed tumor growth and enhanced survival compared with injections into control C57Bl/6J animals. eFABP4 treatment of MCF-7 cells resulted in a significant increase in phosphorylation of extracellular signal-regulated kinase 1/2 (pERK), transcriptional activation of nuclear factor E2-related factor 2 (NRF2) and corresponding gene targets ALDH1A1, CYP1A1, HMOX1, SOD1 and decreased oxidative stress, while R126Q treatment did not show any effects. Proximity-labeling employing an APEX2-FABP4 fusion protein revealed several proteins functioning in desmosomes as eFABP4 receptor candidates including desmoglein (DSG), desmocollin, junction plankoglobin, desomoplankin, and cytokeratins. AlphaFold modeling predicted an interaction between eFABP4, and the extracellular cadherin repeats of DSG2 and pull-down and immunoprecipitation assays confirmed complex formation that was potentiated by oleic acid. Silencing of DSG2 in MCF-7 cells attenuated eFABP4 effects on cellular proliferation, pERK levels, and ALDH1A1 expression compared with controls. IMPLICATIONS: These results suggest desmosomal proteins, and in particular desmoglein 2, may function as receptors of eFABP4 and provide new insight into the development and progression of obesity-associated cancers.