RESUMEN
NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.
Asunto(s)
Adenilato Quinasa/química , Resonancia Magnética Nuclear Biomolecular/métodos , Adenilato Quinasa/análisis , Elementos de la Serie de los Lantanoides/química , Modelos Moleculares , Conformación ProteicaRESUMEN
For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)(2)FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)(2)FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)(2)FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Miosinas/química , Adenosina Trifosfato/química , Dictyostelium/química , Cinética , Conformación Proteica , Espectrometría de FluorescenciaRESUMEN
We have used two complementary time-resolved spectroscopic techniques, dipolar electron-electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF(x)) or ADP.P(i) (ADP.AlF(4)) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF(4) was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.
Asunto(s)
Miosinas/química , Adenosina Difosfato/química , Adenosina Trifosfato/química , Espectroscopía de Resonancia por Spin del Electrón , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Conformación Proteica , Marcadores de SpinRESUMEN
We have engineered a mutant of Dictyostelium discoideum (Dicty) myosin II that contains the same fast-reacting "SH1" thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the force-generating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, (i) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and (ii) these structural states are essentially identical in the two myosins but (iii) are occupied to different extents as a function of the biochemical state. We conclude that (i) myosin structural and biochemical states do not have a one-to-one correspondence, and (ii) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account.
Asunto(s)
Músculos/química , Miosinas/química , Marcadores de Spin , Adenosina Trifosfatasas/metabolismo , Animales , Dictyostelium , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Hidrólisis , Espectrometría de Masas , Músculos/metabolismo , Miosinas/metabolismo , Nucleótidos/química , Nucleótidos/metabolismo , Conejos , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process. Understanding the ternary complex geometry offers valuable insight into selectivity, catalytic efficiency, linker chemistry, and rational degrader design. In this study, we utilize hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify degrader-induced protein-protein interfaces. We then use these data in conjunction with constrained protein docking to build three-dimensional models of the ternary complex. The approach was used to characterize complex formation between the E3 ligase CRBN and the first bromodomain of BRD4, a prominent oncology target. We show marked differences in the ternary complexes formed in solution based on distinct patterns of deuterium uptake for two degraders, CFT-1297 and dBET6. CFT-1297, which exhibited positive cooperativity, altered the deuterium uptake profile revealing the degrader-induced protein-protein interface of the ternary complex. For CFT-1297, the ternary complexes generated by the highest scoring HDX-constrained docking models differ markedly from those observed in the published crystal structures. These results highlight the potential utility of HDX-MS to provide rapidly accessible structural insights into degrader-induced protein-protein interfaces in solution. They further suggest that degrader ternary complexes exhibit significant conformation flexibility and that biologically relevant complexes may well not exhibit the largest interaction surfaces between proteins. Taken together, the results indicate that methods capable of incorporating linker conformation uncertainty may prove an important component in degrader design moving forward. In addition, the development of scoring functions modified to handle interfaces with no evolved complementarity, for example, through consideration of high levels of water infiltration, may prove valuable. Furthermore, the use of crystal structures as validation tools for novel degrader methods needs to be considered with caution.
Asunto(s)
Proteínas de Ciclo Celular/química , Simulación por Computador , Medición de Intercambio de Deuterio , Espectrometría de Masas/métodos , Factores de Transcripción/química , Acetamidas/química , Acetamidas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Indoles/química , Indoles/farmacología , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Piperidinas/química , Piperidinas/farmacología , Conformación ProteicaRESUMEN
Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bioensayo , Proteínas de Ciclo Celular/metabolismo , Oxindoles/farmacología , Ftalimidas/farmacología , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Células HeLa , Humanos , Cinética , Oxindoles/síntesis química , Ftalimidas/síntesis química , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Unión Proteica , Dominios Proteicos , Proteolisis/efectos de los fármacos , Termodinámica , Factores de Transcripción/química , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.
Asunto(s)
Cloroplastos/metabolismo , Óxidos N-Cíclicos/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Imidazolidinas/química , Imidazolinas/química , Marcadores de Spin , Tilacoides/metabolismo , Vicia faba/metabolismo , Adenosina Trifosfato/metabolismo , Calibración , Cloroplastos/efectos de los fármacos , Cloroplastos/efectos de la radiación , Espectroscopía de Resonancia por Spin del Electrón/normas , Concentración de Iones de Hidrógeno , Modelos Biológicos , Modelos Químicos , Oxalatos/farmacología , Fotosíntesis , Bombas de Protones/metabolismo , Fuerza Protón-Motriz , Tilacoides/efectos de los fármacos , Tilacoides/efectos de la radiación , Vicia faba/efectos de los fármacos , Vicia faba/efectos de la radiaciónRESUMEN
Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Modelos Químicos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/ultraestructura , Músculo Esquelético/química , Miosinas/química , Miosinas/ultraestructura , Animales , Simulación por Computador , Elasticidad , Modelos Moleculares , Técnicas de Sonda Molecular , Movimiento (Física) , Conformación Proteica , Conejos , Estrés MecánicoRESUMEN
Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Mesilato de Imatinib/farmacología , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/farmacología , Aurora Quinasa A/química , Aurora Quinasa A/metabolismo , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Humanos , Mesilato de Imatinib/química , Mesilato de Imatinib/metabolismo , Cinética , Unión Proteica , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismoRESUMEN
With early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme's key kinetic obstacle-how to maintain catalytic speed on a cooler Earth-by exploiting transition-state heat capacity. Tracing the evolution of enzyme activity and stability from the hot-start toward modern hyperthermophilic, mesophilic, and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level, refutes the debated activity/stability trade-off, and suggests that the catalytic speed of adenylate kinase is an evolutionary driver for organismal fitness.
Asunto(s)
Adenilil Ciclasas/química , Biocatálisis , Termotolerancia , Adenilil Ciclasas/clasificación , Adenilil Ciclasas/genética , Evolución Molecular , Calor , Cinética , Mutación , FilogeniaRESUMEN
Design of specific kinase inhibitors is an appealing approach for developing new anticancer treatments. However, only a few success stories have been reported to date. Here we demonstrate how the combination of old-fashioned and new biophysical tools together with recent advances in genomics and molecular evolution can aid in overcoming existing limitations.
RESUMEN
Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/química , Recoverina/química , Escherichia coli , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Cuaternaria de Proteína , Recoverina/genética , Recoverina/metabolismoRESUMEN
Sophisticated protein kinase networks, empowering complexity in higher organisms, are also drivers of devastating diseases such as cancer. Accordingly, these enzymes have become major drug targets of the twenty-first century. However, the holy grail of designing specific kinase inhibitors aimed at specific cancers has not been found. Can new approaches in cancer drug design help win the battle with this multi-faced and quickly evolving enemy? In this perspective we discuss new strategies and ideas that were born out of a recent breakthrough in understanding the molecular basis underlying the clinical success of the cancer drug Gleevec. An "old" method, stopped-flow kinetics, combined with old enzymes, the ancestors dating back up to about billion years, provides an unexpected outlook for future intelligent design of drugs.
RESUMEN
Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg(2+) cofactor activates two distinct molecular events: phosphoryl transfer (>10(5)-fold) and lid opening (10(3)-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 10(3)-fold without substantially affecting lid opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.
Asunto(s)
Adenilato Quinasa/química , Adenilato Quinasa/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos MolecularesRESUMEN
Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abl's high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.