RESUMO
Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.
Assuntos
Actinas , Perda Auditiva , Miosina Tipo III , Animais , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Chlorocebus aethiops , Células COS , Perda Auditiva/genética , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Miosina Tipo III/genética , Miosina Tipo III/metabolismo , Miosinas/genética , Miosinas/metabolismo , Estereocílios , HumanosRESUMO
BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.
Assuntos
Miosina Tipo I , Síndrome Nefrótica , Animais , Humanos , Camundongos , Mutação , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Síndrome Nefrótica/genética , EsteroidesRESUMO
Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a "nanosurfer" assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human ß-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0-C2 or C1-C2) on nanotubes every 14 nm. Both C0-C2 and C1-C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0-C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0-C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of ß-cardiac myosin contractility.
Assuntos
Miosinas Cardíacas , Miosinas Ventriculares , Miosinas Cardíacas/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Miocárdio/metabolismo , Miosinas/metabolismo , Fosforilação , Miosinas Ventriculares/análise , Miosinas Ventriculares/metabolismoRESUMO
Myosins are actin-based motor proteins known to perform a variety of different mechanical tasks in cells including transporting cargo, generating tension, and linking the cytoskeleton and membrane. Myosins that function as transporters often form complexes with adaptor proteins and vesicular membranes, making it unclear how they transport their cargo through the actin cytoskeletal network. Rai et al. now use single-molecule kinetics, FRET, and DNA origami scaffolds that mimic motor-adaptor complexes to reveal that the myosin VI-Dab2 complex, which is held together weakly and turns over rapidly, can facilitate processive transport without disruption of the cytoskeleton.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Citoesqueleto de Actina/metabolismo , Transporte Biológico , Proteínas do Citoesqueleto/metabolismo , Humanos , Ligação Proteica , Frações Subcelulares/metabolismoRESUMO
Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human ß-cardiac myosin subfragment 1 (M2ß-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2ß-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2ß-S1. Our results demonstrated M2ß-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2ß-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2ß-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.
Assuntos
Actinas , Cardiomiopatias , Actinas/genética , Trifosfato de Adenosina , Miosinas Cardíacas , Humanos , Mutação , Subfragmentos de MiosinaRESUMO
Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.
Assuntos
Actinas/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Miosina Tipo V/metabolismo , Imagem Óptica/métodos , Fosfatos/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Galinhas , Cinética , Modelos Moleculares , Mutação , Miosina Tipo V/genéticaRESUMO
We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human ß-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human ß-cardiac myosin subfragment 1 (M2ß-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.
Assuntos
Cardiomiopatia Dilatada/genética , Insuficiência Cardíaca/genética , Ureia/análogos & derivados , Miosinas Ventriculares/genética , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Actomiosina/genética , Adenosina Trifosfatases/genética , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/patologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/patologia , Humanos , Cinética , Atividade Motora/genética , Mutação , Contração Miocárdica/efeitos dos fármacos , Domínios Proteicos/genética , Ureia/farmacologia , Miosinas Ventriculares/química , Miosinas Ventriculares/metabolismoRESUMO
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.
Assuntos
Mecanotransdução Celular , Atividade Motora , Mutação , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Modelos Moleculares , Miosina Tipo V/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de SequênciaRESUMO
Actin and myosin play important roles in many devastating diseases and thus are attractive targets for small-molecule therapy. In this issue of JBC, Guhathakurta et al. have developed a high-throughput screening assay to find small molecules that interfere with the actomyosin interaction. They utilized time-resolved FRET (TR-FRET) and a unique donor-acceptor pair (filamentous actin and a peptide that binds near the myosin-binding site on actin) to find novel molecules that interfere with the actomyosin ATPase and alter the structure of actin filaments. These findings demonstrate the power and potential of high-throughput TR-FRET in monitoring molecular interactions.
Assuntos
Actomiosina/antagonistas & inibidores , Actomiosina/química , Actomiosina/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência/métodos , Cinética , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Ligação ProteicaRESUMO
The small molecule drug omecamtiv mecarbil (OM) specifically targets cardiac muscle myosin and is known to enhance cardiac muscle performance, yet its impact on human cardiac myosin motor function is unclear. We expressed and purified human ß-cardiac myosin subfragment 1 (M2ß-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2ß-S1 is slowed more than 4-fold in the presence of OM, whereas the actin concentration required for half-maximal ATPase was reduced dramatically (30-fold). We find OM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence of OM. The dominant pathway results in a slow transition between actomyosin·ADP states and increases the time myosin is strongly bound to actin. However, OM also traps a population of myosin heads in a weak actin affinity state with slow product release. We demonstrate that OM can reduce the actin sliding velocity more than 100-fold in the in vitro motility assay. The ionic strength dependence of in vitro motility suggests the inhibition may be at least partially due to drag forces from weakly attached myosin heads. OM causes an increase in duty ratio examined in the motility assay. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force development (ktr) in a concentration-dependent manner, whereas the maximally activated force is unchanged. We propose that OM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium.
Assuntos
Cálcio/química , Miocárdio/metabolismo , Ureia/análogos & derivados , Miosinas Ventriculares/química , Actinas/química , Actomiosina/química , Difosfato de Adenosina/química , Animais , Relação Dose-Resposta a Droga , Humanos , Cinética , Espectrometria de Massas , Camundongos , Miosinas/química , Domínios Proteicos , Proteínas Recombinantes/química , Estresse Mecânico , Ureia/químicaRESUMO
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.
Assuntos
Miosina Tipo V/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Catálise , Domínio Catalítico , Transferência Ressonante de Energia de FluorescênciaRESUMO
Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.
Assuntos
Actinas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/metabolismo , Pseudópodes/metabolismo , Actinas/genética , Animais , Células COS , Chlorocebus aethiops , Humanos , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/genética , Pseudópodes/genéticaRESUMO
Hereditary hearing loss (HL) is characterized by both allelic and locus genetic heterogeneity. Both recessive and dominant forms of HL may be caused by different mutations in the same deafness gene. In a family with post-lingual progressive non-syndromic deafness, whole-exome sequencing of genomic DNA from five hearing-impaired relatives revealed a single variant, p.Gly488Glu (rs145970949:G>A) in MYO3A, co-segregating with HL as an autosomal dominant trait. This amino acid change, predicted to be pathogenic, alters a highly conserved residue in the motor domain of MYO3A. The mutation severely alters the ATPase activity and motility of the protein in vitro, and the mutant protein fails to accumulate in the filopodia tips in COS7 cells. However, the mutant MYO3A was able to reach the tips of organotypic inner ear culture hair cell stereocilia, raising the possibility of a local effect on positioning of the mechanoelectrical transduction (MET) complex at the stereocilia tips. To address this hypothesis, we investigated the interaction of MYO3A with the cytosolic tail of the integral tip-link protein protocadherin 15 (PCDH15), a core component of MET complex. Interestingly, we uncovered a novel interaction between MYO3A and PCDH15 shedding new light on the function of myosin IIIA at stereocilia tips.
Assuntos
Caderinas/metabolismo , Surdez/genética , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/genética , Miosina Tipo III/metabolismo , Polimorfismo de Nucleotídeo Único , Substituição de Aminoácidos , Animais , Células COS , Proteínas Relacionadas a Caderinas , Células Cultivadas , Criança , Pré-Escolar , Chlorocebus aethiops , Surdez/metabolismo , Feminino , Predisposição Genética para Doença , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, ß-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg(2+)-dependent manner (0.3-9.0 mm free Mg(2+)) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg(2+) in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg(2+) in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg(2+) coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg(2+) concentrations, demonstrating that the ADP release rate constant is slowed by Mg(2+) in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg(2+) reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg(2+) inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg(2+)-dependent alterations in actin binding. Overall, our results suggest that Mg(2+) reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.
Assuntos
Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Magnésio/fisiologia , Proteínas Motores Moleculares/metabolismo , Miosinas/metabolismo , Animais , Cinética , Miocárdio/metabolismo , Ligação Proteica , Coelhos , SuínosRESUMO
Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/metabolismo , Pseudópodes/enzimologia , Actinas/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/química , Miosina Tipo III/genética , Fosforilação , Estrutura Terciária de Proteína , Pseudópodes/genética , Células Sf9 , SpodopteraRESUMO
Inherited mutations in human beta-cardiac myosin (M2ß) can lead to severe forms of heart failure. The E525K mutation in M2ß is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2ß subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 µM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2ß S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.
RESUMO
Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in ß-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super-relaxed" state (SRX), which may be further stabilized by a structural state known as the "interacting heads motif" (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human ß-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.
Assuntos
Miosinas Cardíacas , Cardiomiopatia Dilatada , Miosinas Ventriculares , Animais , Humanos , Actinas/metabolismo , Actinas/genética , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Mutação , Contração Miocárdica/fisiologia , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismoRESUMO
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.