RESUMO
Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, ß-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements.
RESUMO
Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies.
Assuntos
Proteínas de Ligação ao Cálcio , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Espectroscopia de Ressonância de Spin Eletrônica/métodos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Marcadores de Spin , Retículo Endoplasmático/metabolismo , Cálcio/metabolismo , Retículo Sarcoplasmático/metabolismo , MicropeptídeosRESUMO
In cardiomyocytes, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a central component of intracellular Ca2+ regulation. Several heart diseases, including heart failure, are associated with reduced myocardial contraction due to SERCA2a downregulation. Therefore, the need for developing new drugs that could improve SERCA2a function is high. We have recently identified SERCA2a modulators (Compounds 6 and 8) from our screening campaigns and confirmed activation of biochemical SERCA2a ATPase activity and Ca2+ uptake activity. In this study, confocal microscopy and in-cell Ca2+ imaging were used to characterize the effects of these SERCA2a activators on Ca2+ regulation in mouse ventricular myocytes and endoplasmic reticulum (ER) Ca2+ uptake in a HEK293 cell expressing human SERCA2a. Analysis of cytosolic Ca2+ dynamics in cardiomyocytes revealed that both Compounds (6 and 8) increase the action potential-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load. While Compound 6 induced a negligible effect on Ca2+ transients invoked by the L-type Ca2+ channel (LTCC) current, Compound 8 increased Ca2+ transients during LTCC activation, suggesting an off-target protein interaction of Compound 8. Analysis of ER Ca2+ transport by human SERCA2a in HEK cells showed that only Compound 6 increased both ER Ca2+ uptake and ER Ca2+ load significantly, whereas Compound 8 had no effect on SERCA2a Ca2+ transport. This study revealed that Compound 6 exhibits promising characteristics that can improve intracellular Ca2+ dynamics by selectively enhancing SERCA2a Ca2+ uptake.
Assuntos
Sinalização do Cálcio , Cálcio , Camundongos , Humanos , Animais , Cálcio/metabolismo , Células HEK293 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.
Assuntos
Actinas/química , Actinas/metabolismo , Miosinas Cardíacas/metabolismo , Actinas/efeitos dos fármacos , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Miosinas Cardíacas/efeitos dos fármacos , Miosinas Cardíacas/fisiologia , Bovinos , Fluorescência , Ensaios de Triagem em Larga Escala/métodos , Cinética , Contração Muscular/fisiologia , Subfragmentos de Miosina/efeitos dos fármacos , Subfragmentos de Miosina/metabolismo , Miosinas/efeitos dos fármacos , Miosinas/metabolismo , Física , Ligação Proteica , Pirenos/química , Coelhos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Benzilaminas/farmacologia , Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica Familiar/tratamento farmacológico , Subfragmentos de Miosina/metabolismo , Uracila/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Benzilaminas/uso terapêutico , Miosinas Cardíacas/química , Cardiomiopatia Hipertrófica Familiar/etiologia , Humanos , Cinética , Subfragmentos de Miosina/química , Estabilidade Proteica/efeitos dos fármacos , Uracila/farmacologia , Uracila/uso terapêuticoRESUMO
We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.
Assuntos
Actomiosina/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Mutação , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Actinas/química , Actinas/metabolismo , Actomiosina/química , Actomiosina/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Miosinas Cardíacas/química , Bovinos , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/química , CoelhosRESUMO
We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCAâ™s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca 2+ -ATPase and Ca 2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.
RESUMO
We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCAâ™s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca 2+ -ATPase and Ca 2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.
RESUMO
We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA's function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Insuficiência Cardíaca , Humanos , Ensaios de Triagem em Larga Escala , Coração , Insuficiência Cardíaca/tratamento farmacológico , Adenosina TrifosfatasesRESUMO
The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+-uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.
Assuntos
ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Retículo Sarcoplasmático , Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/metabolismo , Transporte de Íons , Retículo Endoplasmático/metabolismo , Células Musculares/metabolismo , Cálcio/metabolismo , Mamíferos/metabolismoRESUMO
Familial hypertrophic cardiomyopathy is a disease caused by single mutations in several sarcomeric proteins, including the human myosin ventricular regulatory light chain (vRLC). The effects of four of these mutations (A13T, F18L, E22K, and P95A) in vRLC on force generation were determined as a function of Ca(2+) concentration. The endogenous RLC was removed from skinned rabbit psoas muscle fibers, and replaced with either rat wildtype vRLC or recombinant rat vRLC (G13T, F18L, E22K, and P95A). Compared to fibers with wildtype rat vRLC, the E22K mutant increased Ca sensitivity of force generation, whereas the G13T and F18L mutants decreased the Ca sensitivity, and the P95A mutant had no significant effect. None of the RLC mutants affected the maximal tension (observed at saturating Ca(2+) concentrations), except for F18L, which decreased the maximal tension to 69 +/- 10% of the wildtype value. Of the mutant RLCs, only F18L decreased the cooperativity of activation of force generation. These results suggest that the primary cause of familial hypertrophic cardiomyopathy, in some cases, is perturbation in the Ca sensitivity of force generation, in which Ca-sensitizing or Ca-desensitizing effects can lead to similar disease phenotypes.
Assuntos
Cálcio/metabolismo , Cardiomiopatia Hipertrófica Familiar/fisiopatologia , Contração Isométrica , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Músculos Psoas/fisiopatologia , Animais , Cálcio/farmacologia , Cardiomiopatia Hipertrófica Familiar/genética , Cardiomiopatia Hipertrófica Familiar/metabolismo , Relação Dose-Resposta a Droga , Coração/fisiopatologia , Humanos , Técnicas In Vitro , Mutação , Miocárdio/metabolismo , Coelhos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse MecânicoRESUMO
Figure 3 summarizes the effects of actomyosin binding on the internal and global dynamics of either protein, as discussed in this chapter. These effects depend primarily on the strength of the interaction; which in turn depends on the state of the nucleotide at the myosin active site. When either no nucleotide or ADP is bound, the interaction is strong and the effect on each protein is maximal. When the nucleotide is ATP or ADP.Pi, or the equivalent nonhydrolyzable analogs, the interaction is weak and the effect on molecular dynamics of each protein is minimal. The weaker effects in weak-binding states are not simply the reflection of lower occupancy of binding sites--the molecular models in Fig. 3 illustrate the effects of the formation of the ternary complex, after correction for the free actin and myosin in the system. Thus EPR on myosin (Berger and Thomas 1991; Thomas et al. 1995) and pyrene fluorescence studies on actin (Geeves 1991) have shown that the formation of a ternary complex has a negligible effect on the internal dynamics of both [figure: see text] proteins (left side of Fig. 3, white arrows). As shown by both EPR (Baker et al. 1998; Roopnarine et al. 1998) and phosphorescence (Ramachandran and Thomas 1999), both domains of myosin are dynamically disordered in weak-binding states, and this is essentially unaffected by the formation of the ternary complex (left side of Fig. 3, indicated by disordered myosin domains). The only substantial effect of the formation of the weak interaction that has been reported is the EPR-detected (Ostap and Thomas 1991) restriction of the global dynamics of actin upon weak myosin binding (left column of Fig. 3, gray arrow). The effects of strong actomyosin formation are much more dramatic. While substantial rotational dynamics, both internal and global, exist in both myosin and actin in the presence of ADP or the absence of nucleotides, spin label EPR, pyrene fluorescence, and phosphorescence all show dramatic restrictions in these motions upon formation of the strong ternary complex (right column of Fig. 3). One implication of this is that the weak-to-strong transition is accompanied by a disorder-to-order transition in both actin and myosin, and this is itself an excellent candidate for the structural change that produces force (Thomas et al. 1995). Another clear implication is that the crystal structures obtained for isolated myosin and actin are not likely to be reliable representations of structures that exist in ternary complexes of these proteins (Rayment et al. 1993a and 1993b; Dominguez et al. 1998; Houdusse et al. 1999). This is clearly true of the strong-binding states, since the spectroscopic studies indicate consistently that substantial changes occur in both proteins upon strong complex formation. For the weak complexes, the problem is not that complex formation induces large structural changes, but that the structures themselves are dynamically disordered. This is probably why so many different structures have been obtained for myosin S1 with nucleotides bound--each crystal is selecting one of the many different substates represented by the dynamic ensemble. Finally, there is the problem that the structures of actomyosin complexes are probably influenced strongly by their mechanical coupling to muscle protein lattice (Baker at al. 2000). Thus, even if co-crystals of actin and myosin are obtained in the future, an accurate description of the structural changes involved in force generation will require further experiments using site-directed spectroscopic probes of both actin and myosin, in order to detect the structural dynamics of these ternary complexes under physiological conditions.