RESUMEN
Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best-suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7-fold higher molecular brightness and 6.4-fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant.
Asunto(s)
Calcio , Troponina C , Humanos , Animales , Troponina C/genética , Troponina C/química , Troponina C/metabolismo , Calcio/metabolismo , Proteínas Fluorescentes Verdes/química , Células HeLa , Neuronas/metabolismo , MamíferosRESUMEN
Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).
Asunto(s)
Cardiomiopatías , Cardiomiopatía Dilatada , Ratones , Humanos , Animales , Troponina C/genética , Troponina C/química , Troponina C/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Mutación , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Contracción Miocárdica , Calcio/metabolismoRESUMEN
Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM.
Asunto(s)
Músculo Estriado , Troponina C , Troponina C/química , Calcio/metabolismo , Músculo Estriado/metabolismo , Relación Estructura-ActividadRESUMEN
Calcium-binding proteins play critical roles in various biological processes such as signal transduction, cell growth, and transcription factor regulation. Ion binding and target binding of Ca2+-binding proteins are highly related. Therefore, understanding the ion binding mechanism will benefit the relevant inhibitor design toward the Ca2+-binding proteins. The EF-hand is the typical ion binding motif in Ca2+-binding proteins. Previous studies indicate that the ion binding affinity of the EF-hand increases with the peptide length, but this mechanism has not been fully understood. Herein, using molecular dynamics simulations, thermodynamic integration calculations, and molecular mechanics Poisson-Boltzmann surface area analysis, we systematically investigated four Ca2+-binding peptides containing the EF-hand loop in site III of rabbit skeletal troponin C. These four peptides have 13, 21, 26, and 34 residues. Our simulations reproduced the observed trend that the ion binding affinity increases with the peptide length. Our results implied that the E-helix motif preceding the EF-hand loop, likely the Phe99 residue in particular, plays a significant role in this regulation. The E-helix has a significant impact on the backbone and side-chain conformations of the Asp103 residue, rigidifying important hydrogen bonds in the EF-hand and decreasing the solvent exposure of the Ca2+ ion, hence leading to more favorable Ca2+ binding in longer peptides. The present study provides molecular insights into the ion binding in the EF-hand and establishes an important step toward elucidating the responses of Ca2+-binding proteins toward the ion and target availability.
Asunto(s)
Simulación de Dinámica Molecular , Troponina C , Animales , Conejos , Troponina C/química , Calcio/metabolismo , Estructura Terciaria de Proteína , Péptidos/química , Sitios de Unión , Unión ProteicaRESUMEN
NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.
Asunto(s)
Calcio , Troponina C , Animales , Calcio/metabolismo , Señalización del Calcio , Calmodulina/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Indicadores y Reactivos , Troponina C/genética , Troponina C/química , Troponina C/metabolismoRESUMEN
Calcium-dependent heart muscle contraction is regulated by the cardiac troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). cNTnC contains one calcium binding site (site II), and altered calcium binding in this site has been studied for decades. It has been previously shown that cNTnC mutants, which increase calcium sensitization may have therapeutic benefits, such as restoring cardiac muscle contractility and functionality post-myocardial infarction events. Here, we computationally characterized eight mutations for their potential effects on calcium binding affinity in site II of cNTnC. We utilized two distinct methods to estimate calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic integration (TI). We observed a sensitizing trend for all mutations based on the employed ASMD methodology. The TI results showed excellent agreement with experimentally known calcium binding affinities in wild-type cNTnC. Based on the TI results, five mutants were predicted to increase calcium sensitivity in site II. This study presents an interesting comparison of the two computational methods, which have both been shown to be valuable tools in characterizing the impacts of calcium sensitivity in mutant cNTnC systems.
Asunto(s)
Calcio , Troponina C , Troponina C/química , Calcio/metabolismo , Troponina I/metabolismo , Unión Proteica , Sitios de UniónRESUMEN
The cardiac troponin (cTn) complex is an important regulatory protein in heart contraction. Upon binding of Ca2+, cTn undergoes a conformational shift that allows the troponin I switch peptide (cTnISP) to be released from the actin filament and bind to the troponin C hydrophobic patch (cTnCHP). Mutations and modifications to this complex can change its sensitivity to Ca2+ and alter the energetics of the transition from the Ca2+-unbound, cTnISP-unbound form to the Ca2+-bound, cTnISP-bound form. We utilized targeted molecular dynamics (TMD) to obtain a trajectory of this transition pathway, followed by umbrella sampling to estimate the free energy associated with the cTnISP-cTnCHP binding and the cTnCHP opening events for wild-type (WT) cTn. We were able to reproduce experimental values for the cTnISP-cTnCHP binding event and obtain cTnCHP opening free energies in agreement with previous computational measurements of smaller cTnC systems. This excellent agreement for WT cTn demonstrated the strength of computational methods in studying the dynamics and energetics of the cTn complex. We then introduced mutations to the cTn complex that cause cardiomyopathy or alter its Ca2+ sensitivity and observed a general decrease in the free energy of opening the cTnCHP. For these same mutations, we observed no general trend in the effect on the cTnISP-cTnCHP binding event. Our method sets the stage for future computational studies on this system that predict the consequences of yet uncharacterized mutations on cTn dynamics and energetics.
Asunto(s)
Calcio , Troponina C , Calcio/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Troponina C/química , Troponina I/metabolismoRESUMEN
W7 is a sarcomere inhibitor that decreases the calcium sensitivity of force development in cardiac muscle. W7 binds to the interface of the regulatory domain of cardiac troponin C (cNTnC) and the switch region of troponin I (cTnI), decreasing the binding of cTnI to cNTnC, presumably by electrostatic repulsion between the -NH3+ group of W7 and basic amino acids in cTnI. W7 analogs with a -CO2- tail are inactive. To evaluate the importance of the location of the charged -NH3+, we used a series of compounds W4, W6, W8, and W9, which have three less, one less, one more, and two more methylene groups in the tail region than W7. W6, W8, and W9 all bind tighter to cNTnC-cTnI chimera (cChimera) than W7, while W4 binds weaker. W4 and, strikingly, W6 have no effect on calcium sensitivity of force generation, while W8 and W9 decrease calcium sensitivity, but less than W7. The structures of the cChimera-W6 and cChimera-W8 complexes reveal that W6 and W8 bind to the same hydrophobic cleft as W7, with the aliphatic tail taking a similar route to the surface. NMR relaxation data show that internal flexibility in the tail of W7 is very limited. Alignment of the cChimera-W7 structure with the recent cryoEM structures of the cardiac sarcomere in the diastolic and systolic states reveals the critical location of the amino group. Small molecule induced structural changes can therefore affect the tightly balanced equilibrium between tethered components required for rapid contraction.
Asunto(s)
Sarcómeros , Troponina C , Calcio/metabolismo , Miocardio/metabolismo , Sarcómeros/metabolismo , Sulfonamidas/química , Troponina C/química , Troponina I/químicaRESUMEN
The troponin core is an important regulatory complex in cardiac sarcomeres. Contraction is initiated by a calcium ion binding to cardiac troponin C (cTnC), initiating a conformational shift within the protein, altering its interactions with cardiac troponin I (cTnI). The change in cTnC-cTnI interactions prompts the C-terminal domain of cTnI to dissociate from actin, allowing tropomyosin to reveal myosin-binding sites on actin. Each of the concerted movements in the cardiac thin filament (CTF) is crucial for allowing the contraction of cardiomyocytes, yet little is known about the free energy associated with each transition, which is vital for understanding contraction on a molecular level. Using metadynamics, we calculated the free-energy surface of two transitions in the CTF: cTnC opening in the presence and absence of Ca2+ and cTnI dissociating from actin with both open and closed cTnC. These results not only provide the free-energy surface of the transitions but will also be shown to determine if the order of transitions in the contraction cycle is important. From our calculations, we found that the calcium ion helps stabilize the open conformation of cTnC and that the C-terminus of cTnI is stabilized by cTnC in the open conformation when dissociating from the actin surface.
Asunto(s)
Sarcómeros , Troponina C , Actinas/metabolismo , Calcio/química , Contracción Muscular , Miocardio/metabolismo , Sarcómeros/metabolismo , Troponina C/química , Troponina I/metabolismoRESUMEN
The C-terminal 14-16 residues of human troponin T are required for full inactivation, and they prevent full activation at saturating Ca2+. Basic residues within that C-terminal region of TnT are essential for its function, but the mechanism of action is unknown. That region of TnT is natively disordered and does not appear in reconstructions of the troponin structure. We used Förster resonance energy transfer to determine if the C-terminal basic region of TnT alters transitions of TnI or if it operates independently. We also examined Ca2+-dependent changes in the C-terminal region of TnT itself. Probes on TnI-143 (inhibitory region) and TnI-159 (switch region) moved away from sites on actin and tropomyosin and toward TnC-84 at high Ca2+. Ca2+ also displaced C-terminal TnT from actin-tropomyosin but without movement toward TnC. Deletion of C-terminal TnT produced changes in TnI-143 like those effected by Ca2+, but effects on TnI-159 were muted; there was no effect on the distance of the switch region to TnC-84. Substituting Ala for basic residues within C-terminal TnT displaced C-terminal TnT from actin-tropomyosin. The results suggest that C-terminal TnT stabilizes tropomyosin in the inactive position on actin. Removal of basic residues from C-terminal TnT produced a Ca2+-like state except that the switch region of TnI was not bound to TnC. Addition of Ca2+ caused more extreme displacement from actin-tropomyosin as the active state became more fully occupied as in the case of wild-type TnT in the presence of both Ca2+ and bound rigor myosin S1.
Asunto(s)
Troponina I , Troponina T , Actinas/metabolismo , Calcio/metabolismo , Humanos , Músculo Esquelético/metabolismo , Tropomiosina/química , Troponina C/química , Troponina C/genética , Troponina I/química , Troponina T/química , Troponina T/genéticaRESUMEN
The X-ray crystal structure of a human cardiac muscle troponin C/troponin I chimera has been determined in two different crystal forms and shows a conformation of the complex that differs from that previously observed by NMR. The chimera consists of the N-terminal domain of troponin C (cTnC; residues 1-80) fused to the switch region of troponin I (cTnI; residues 138-162). In both crystal forms, the cTnI residues form a six-turn α-helix that lays across the hydrophobic groove of an adjacent cTnC molecule in the crystal structure. In contrast to previous models, the cTnI helix runs in a parallel direction relative to the cTnC groove and completely blocks the calcium desensitizer binding site of the cTnC-cTnI interface.
Asunto(s)
Troponina C , Troponina I , Calcio/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Miocardio/química , Unión Proteica , Proteínas Recombinantes de Fusión/química , Troponina C/análisis , Troponina C/química , Troponina I/análisis , Troponina I/químicaRESUMEN
Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the "active" orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR 15N R2 relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R2 10 s-1), being connected by a flexible linker. However, upon addition of cTnI1-77, the complex tumbles as a rigid unit (R2 30 s-1). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate 15N R2 rates (R2 17-23 s-1). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI1-37 and cTnI135-209. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI38-134, cTnC-cTnT223-288, or cTnC-cTnI38-134-cTnT223-288), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a "dormant" orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.
Asunto(s)
Cardiomiopatía Dilatada/genética , Mutación , Miocardio/metabolismo , Troponina C/genética , Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Tropomiosina/química , Tropomiosina/metabolismo , Troponina C/química , Troponina C/metabolismo , Troponina I/química , Troponina I/metabolismo , Troponina T/química , Troponina T/metabolismoRESUMEN
Troponin regulates the calcium-mediated activation of skeletal muscle. Muscle weakness in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy occurs from diminished neuromuscular output. The first direct fast skeletal troponin activator, tirasemtiv, amplifies the response of muscle to neuromuscular input. Tirasemtiv binds selectively and strongly to fast skeletal troponin, slowing the rate of calcium release and sensitizing muscle to calcium. We report the solution NMR structure of tirasemtiv bound to a fast skeletal troponin C-troponin I chimera. The structure reveals that tirasemtiv binds in a hydrophobic pocket between the regulatory domain of troponin C and the switch region of troponin I, which overlaps with that of Anapoe in the X-ray structure of skeletal troponin. Multiple interactions stabilize the troponin C-troponin I interface, increase the affinity of troponin C for the switch region of fast skeletal troponin I, and drive the equilibrium toward the active state.
Asunto(s)
Imidazoles/farmacología , Músculo Esquelético/efectos de los fármacos , Pirazinas/farmacología , Troponina C/metabolismo , Troponina I/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Imidazoles/química , Simulación del Acoplamiento Molecular , Músculo Esquelético/fisiología , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica/efectos de los fármacos , Pirazinas/química , Troponina C/química , Troponina I/químicaRESUMEN
Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients' biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients' myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.
Asunto(s)
Calcio , Simulación de Dinámica Molecular , Contracción Muscular , Miotonía Congénita , Sarcómeros , Troponina C , Sitios de Unión , Calcio/química , Calcio/metabolismo , Humanos , Miotonía Congénita/genética , Miotonía Congénita/metabolismo , Sarcómeros/química , Sarcómeros/genética , Sarcómeros/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismoRESUMEN
Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.
Asunto(s)
Calcio/metabolismo , Magnesio/metabolismo , Troponina C/metabolismo , Sitios de Unión , Cationes Bivalentes/metabolismo , Humanos , Miocardio/metabolismo , Unión Proteica , Termodinámica , Troponina C/químicaRESUMEN
One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k-Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k-Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SUMMARY: A challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.
Asunto(s)
Modelos Biológicos , Mutación , Contracción Miocárdica , Miofibrillas/genética , Miofibrillas/metabolismo , Troponina C/genética , Algoritmos , Alelos , Animales , Biomarcadores , Calcio/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Miofibrillas/química , Unión Proteica , Sarcómeros/metabolismo , Relación Estructura-Actividad , Troponina C/química , Troponina I/genética , Troponina I/metabolismoRESUMEN
Understanding the regulation of cardiac muscle contraction at a molecular level is crucial for the development of therapeutics for heart conditions. Despite the availability of atomic structures of the protein components of cardiac muscle thin filaments, detailed insights into their dynamics and response to calcium are yet to be fully depicted. In this study, we used molecular dynamics simulations of the core domains of the cardiac muscle protein troponin to characterize the equilibrium dynamics of its calcium-bound and calcium-free forms, with a focus on elements of cardiac muscle contraction activation and deactivation, that is, calcium binding to the cardiac troponin Ca2+ -binding subunit (TnC) and the release of the switch region of the troponin inhibitory subunit (TnI) from TnC. The process of calcium binding to the TnC binding site is described as a three-step process commencing with calcium capture by the binding site residues, followed by cooperative residue interplay bringing the calcium ion to the binding site, and finally, calcium-water exchange. Furthermore, we uncovered a set of TnC-TnI interdomain interactions that are critical for TnC N-lobe hydrophobic pocket dynamics. Absence of these interactions allows the closure of the TnC N-lobe hydrophobic pocket while the TnI switch region remains expelled, whereas if the interactions are maintained, the hydrophobic pocket remains open. Modification of these interactions may fine-tune the ability of the TnC N-lobe hydrophobic pocket to close or remain open, modulate cardiac contractility and present potential therapy-relevant targets.
Asunto(s)
Calcio , Troponina C , Calcio/metabolismo , Simulación de Dinámica Molecular , Transducción de Señal , Troponina C/química , Troponina C/metabolismo , Troponina I/química , Troponina I/metabolismoRESUMEN
Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at <10 nM free Ca2+. 251C-HAHA-TnT and Δ16-TnT mutants greatly increased ATPase activation at 0.2 mM Ca2+, even without high-affinity cross-bridge binding. They also shifted the force-pCa curve of muscle fibers to lower Ca2+ by 0.8-1.2 pCa units (the larger shift for 251C-HAHA-TnT). Shifts in force-pCa were maintained in the presence of para-aminoblebbistatin. The effects of modification of the C-terminal region of TnT on the kinetics of S1 binding to actin were somewhat different from those observed earlier with the cardiac analogue. In general, the C-terminal region of human skeletal TnT is critical to regulation, just as it is in the cardiac system, and is a potential target for modulating activity.
Asunto(s)
Calcio/farmacología , Troponina T/metabolismo , Humanos , Cinética , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Tropomiosina/química , Tropomiosina/metabolismo , Troponina C/química , Troponina C/metabolismo , Troponina I/química , Troponina I/metabolismo , Troponina T/químicaRESUMEN
Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca2+ and to eliminate one of the inactive states at low Ca2+. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca2+, full activation with Δ14-TnT·A8V-TnC decreased the apparent KM for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca2+ with Δ14-TnT; the maximum shortening speed at high Ca2+ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca2+ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca2+ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca2+, elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.
Asunto(s)
Actinas/metabolismo , Calcio/metabolismo , Movimiento Celular , Miocardio/metabolismo , Tropomiosina/metabolismo , Troponina C/metabolismo , Troponina T/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Bovinos , Humanos , Miocardio/citología , Unión Proteica , Troponina C/química , Troponina C/genética , Troponina T/química , Troponina T/genéticaRESUMEN
Heart muscle contraction is regulated by calcium binding to cardiac troponin C. This induces troponin I (cTnI) switch region binding to the regulatory domain of troponin C (cNTnC), pulling the cTnI inhibitory region off actin and triggering muscle contraction. Small molecules targeting this cNTnC-cTnI interface have potential in the treatment of heart disease. Most of these have an aromatic core which binds to the hydrophobic core of cNTnC, and a polar and often charged 'tail'. The calmodulin antagonist W7 is unique in that it acts as calcium desensitizer. W7 binds to the interface of cNTnC and cTnI switch region and weakens cTnI binding, possibly by electrostatic repulsion between the positively charged terminal amino group of W7 and the positively charged RRVR144-147 region of cTnI. To evaluate the role of electrostatics, we synthesized A7, where the amino group of W7 was replaced with a carboxyl group. We determined the high-resolution solution NMR structure of A7 bound to a cNTnC-cTnI chimera. The structure shows that A7 does not change the overall conformation of the cNTnC-cTnI interface, and the naphthalene ring of A7 sits in the same hydrophobic pocket as that of W7, but the charged tail takes a different route to the surface of the complex, especially with respect to the position of the switch region of cTnI. We measured the affinities of A7 for cNTnC and the cNTnC-cTnI complex and that of the cTnI switch peptide for the cNTnC-A7 complex. We also compared the binding of W7 and A7 for two cNTnC-cTnI chimeras, differing in the presence or absence of the RRVR region of cTnI. A7 decreased the binding affinity of cTnI to cNTnC substantially less than W7 and bound more tightly to the more positively charged chimera. We tested the effects of W7 and A7 on the force-calcium relation of demembranated rat right ventricular trabeculae and demonstrated that A7 has a much weaker desensitization effect than W7. We also synthesized A6, which has one less methylene group on the hydrocarbon chain than A7. A6 did not affect binding of cTnI switch peptide nor change the calcium sensitivity of ventricular trabeculae. These results suggest that the negative inotropic effect of W7 may result from a combination of electrostatic repulsion and steric hindrance with cTnI.