Direct detection of the myosin super-relaxed state and interacting-heads motif in solution
2021; Elsevier BV; Volume: 297; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2021.101157
ISSN1083-351X
AutoresSami Chu, Joseph M. Muretta, David D. Thomas,
Tópico(s)Lubricants and Their Additives
ResumoThe interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state. The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state. Myosin II is a motor protein that powers the contraction of muscle through large structural changes when its ATPase activity is activated by actin (1Sweeney H.L. Houdusse A. Structural and functional insights into the Myosin motor mechanism.Annu. Rev. Biophys. 2010; 39: 539-557Crossref PubMed Scopus (294) Google Scholar). It consists of a dimer of heavy chain molecules, each binding two light chains, the essential light chain, and regulatory light chain (RLC). In striated muscles (skeletal and cardiac), myosin polymerizes to form bipolar thick filaments through coiled-coil interactions of the C-terminal tail region of the heavy chain. The N-terminal region branches from the tail to form two heads, each containing a globular catalytic domain and a light-chain domain that binds essential light chain and RLC (Fig. 1A).The regulation of muscle contraction involves primarily the calcium-dependent regulation of the actin-containing thin filament. However, an additional regulatory mechanism has recently come to light, in which myosin regulates itself through autoinhibitory interactions between its two heads. It is proposed that the structural basis for this autoinhibited state is the interacting-heads motif (IHM) (2Woodhead J.L. Zhao F.Q. Craig R. Egelman E.H. Alamo L. Padron R. Atomic model of a myosin filament in the relaxed state.Nature. 2005; 436: 1195-1199Crossref PubMed Scopus (229) Google Scholar, 3Lee K.H. Sulbaran G. Yang S. Mun J.Y. Alamo L. Pinto A. Sato O. Ikebe M. Liu X. Korn E.D. Sarsoza F. Bernstein S.I. Padron R. Craig R. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E1991-E2000Crossref PubMed Scopus (44) Google Scholar, 4Alamo L. Pinto A. Sulbaran G. Mavarez J. Padron R. Lessons from a tarantula: New insights into myosin interacting-heads motif evolution and its implications on disease.Biophys. Rev. 2018; 10: 1465-1477Crossref PubMed Scopus (21) Google Scholar, 5Jung H.S. Komatsu S. Ikebe M. Craig R. Head-head and head-tail interaction: A general mechanism for switching off myosin II activity in cells.Mol. Biol. Cell. 2008; 19: 3234-3242Crossref PubMed Scopus (133) Google Scholar). Evidence for the IHM has been obtained in several isoforms of myosin from several species through electron microscopy (3Lee K.H. Sulbaran G. Yang S. Mun J.Y. Alamo L. Pinto A. Sato O. Ikebe M. Liu X. Korn E.D. Sarsoza F. Bernstein S.I. Padron R. Craig R. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E1991-E2000Crossref PubMed Scopus (44) Google Scholar, 5Jung H.S. Komatsu S. Ikebe M. Craig R. Head-head and head-tail interaction: A general mechanism for switching off myosin II activity in cells.Mol. Biol. Cell. 2008; 19: 3234-3242Crossref PubMed Scopus (133) Google Scholar, 6Zoghbi M.E. Woodhead J.L. Moss R.L. Craig R. Three-dimensional structure of vertebrate cardiac muscle myosin filaments.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2386-2390Crossref PubMed Scopus (184) Google Scholar, 7Xu J.Q. Harder B.A. Uman P. Craig R. Myosin filament structure in vertebrate smooth muscle.J. Cell Biol. 1996; 134: 53-66Crossref PubMed Scopus (89) Google Scholar), and evidence consistent with it has been observed by X-ray diffraction (8Linari M. Brunello E. Reconditi M. Fusi L. Caremani M. Narayanan T. Piazzesi G. Lombardi V. Irving M. Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.Nature. 2015; 528: 276-279Crossref PubMed Scopus (162) Google Scholar, 9Caremani M. Pinzauti F. Powers J.D. Governali S. Narayanan T. Stienen G.J.M. Reconditi M. Linari M. Lombardi V. Piazzesi G. Inotropic interventions do not change the resting state of myosin motors during cardiac diastole.J. Gen. Physiol. 2019; 151: 53-65Crossref PubMed Scopus (20) Google Scholar) and fluorescence polarization (10Fusi L. Brunello E. Yan Z. Irving M. Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle.Nat. Commun. 2016; 7: 13281Crossref PubMed Scopus (68) Google Scholar, 11Brunello E. Fusi L. Ghisleni A. Park-Holohan S.J. Ovejero J.G. Narayanan T. Irving M. Myosin filament-based regulation of the dynamics of contraction in heart muscle.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 8177-8186Crossref PubMed Scopus (47) Google Scholar). In the IHM, the catalytic domains of the myosin dimer interact asymmetrically—the converter domain of the free head blocks the actin-binding region of the blocked head (3Lee K.H. Sulbaran G. Yang S. Mun J.Y. Alamo L. Pinto A. Sato O. Ikebe M. Liu X. Korn E.D. Sarsoza F. Bernstein S.I. Padron R. Craig R. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E1991-E2000Crossref PubMed Scopus (44) Google Scholar). Another region of interest in the IHM is the mesa region of the catalytic domain, which is proposed to interact with the portion of the tail region closest to the catalytic domain (also called subfragment-2 or S2) (12Woodhead J.L. Craig R. The mesa trail and the interacting heads motif of myosin II.Arch. Biochem. Biophys. 2020; 680: 108228Crossref PubMed Scopus (9) Google Scholar, 13Nag S. Trivedi D.V. Sarkar S.S. Adhikari A.S. Sunitha M.S. Sutton S. Ruppel K.M. Spudich J.A. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Nat. Struct. Mol. Biol. 2017; 24: 525-533Crossref PubMed Scopus (94) Google Scholar). Studies have shown that disease-related mutations in this region decrease the affinity of the catalytic domain for S2, thus destabilizing the IHM (13Nag S. Trivedi D.V. Sarkar S.S. Adhikari A.S. Sunitha M.S. Sutton S. Ruppel K.M. Spudich J.A. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Nat. Struct. Mol. Biol. 2017; 24: 525-533Crossref PubMed Scopus (94) Google Scholar). The present study uses a cleaved two-headed version of myosin, heavy meromyosin (HMM), which includes the S2 portion of the tail that stabilizes the dimer but lacks the backbone and other accessory proteins that may further stabilize the IHM.It has been hypothesized that the IHM is the structural basis for the super-relaxed state (SRX), a functional state characterized by extremely slow ATP turnover, 100 times slower than during contraction, and three to ten times slower than is typical for most of the myosin molecules in relaxed muscle (14Hooijman P. Stewart M.A. Cooke R. A new state of cardiac myosin with very slow ATP turnover: A potential cardioprotective mechanism in the heart.Biophys. J. 2011; 100: 1969-1976Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 15Stewart M.A. Franks-Skiba K. Chen S. Cooke R. Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 430-435Crossref PubMed Scopus (139) Google Scholar). SRX is detected biochemically in single-turnover experiments, measuring the decrease in fluorescence that occurs when fluorescent mant-ATP is hydrolyzed, released from myosin, and displaced by nonfluorescent ATP, resulting in a multiple exponential decay, in which the slowest rate (longest lifetime τ) is assigned to the SRX state (14Hooijman P. Stewart M.A. Cooke R. A new state of cardiac myosin with very slow ATP turnover: A potential cardioprotective mechanism in the heart.Biophys. J. 2011; 100: 1969-1976Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This kinetic signature has also been observed in isolated myosin molecules and fragments (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). It is generally assumed that the SRX biochemical kinetic state corresponds to the IHM structural state, but this has not been tested directly on the same sample under similar conditions.To bridge this gap, we have used fluorescence resonance energy transfer (FRET) between the two heads of myosin to detect the IHM structural state directly in solution and directly compare the fraction of myosin molecules in SRX measured by ATP turnover, under similar conditions. In contrast to intensity-based FRET measurement, time-resolved FRET can resolve multiple distinct populations of protein conformations, each giving rise to a distinct lifetime τ of decay after an exciting pulse. To verify that FRET is detecting the IHM, we used a chemical cross-linker that is known to stabilize the IHM.To further evaluate the relationship between IHM and SRX, we compared how both the fluorescent nucleotide turnover and the structural FRET measurement were affected by mavacamten, a small molecule that has completed phase III clinical trials for treating hypertrophic cardiomyopathy and has been shown to increase the SRX population (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar, 17Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human beta-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (125) Google Scholar). Given the current state of knowledge, we expected that mavacamten would increase FRET by increasing the fraction of myosin in IHM. We also tested a range of ionic strength shown to affect the SRX population to further probe the relationship between the IHM and SRX (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar).ResultsFRET labels have no effect on myosin function in the presence of actinPurified bovine cardiac HMM myosin was labeled with EDANS and dabcyl through the exchange of RLC labeled on the C-lobe by sulfhydryl reaction between an exogenous cysteine residue on the protein and maleimide on the label, as described in our previous work (Fig. 1A) (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). To minimize the donor-only population, the ratio of donor to acceptor was 1:3, leading to detectable FRET (Fig. 2A). Labeling did not significantly affect the actin association and disassociation (Fig. 1B), actin-activated ATPase activity (Fig. 1C), or single nucleotide turnover kinetics (Fig. 1D).Figure 2TR-FRET detects the interacting-heads motif, and treatment with glutaraldehyde (GA) increases FRET. Representative waveforms for donor-only (D) and donor–acceptor (DA) samples are shown (A) without GA treatment and (B) after GA treatment. (C) Effect of GA treatment on FRET efficiency (E, as calculated from amplitude-weighted average lifetimes τ of the D and DA waveforms), n = 3 per condition. (D) Changes in the mole fraction of the population participating in FRET.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Time-resolved FRET detects the closed state of HMM myosinTime-resolved fluorescence of labeled HMM was best fit by a two-component model: 63% (±5%) of the donors were unaffected by FRET, having a lifetime identical to the donor-only sample, whereas the remaining donors showed substantial FRET, fitting to a Gaussian distance distribution having a center of 2.0 nm (±0.02) with a full-width of 1.4 nm (±0.02). We conclude that myosin molecules with the open conformation have a donor–acceptor distance that is too large to detect and that 43 ± 5% of the myosin molecules are in the IHM state (Fig. 2D). To verify that we are detecting the IHM, we used a variety of myosin effectors known to increase the population of either IHM or SRX myosin.The closed-state population increases with the trapping of IHM myosin by cross-linkerTo test whether the FRET pair on opposite heads of HMM can measure IHM, labeled HMM was treated with glutaraldehyde, a cross-linker used to trap the IHM in electron microscopy studies (Fig. 2B) (3Lee K.H. Sulbaran G. Yang S. Mun J.Y. Alamo L. Pinto A. Sato O. Ikebe M. Liu X. Korn E.D. Sarsoza F. Bernstein S.I. Padron R. Craig R. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E1991-E2000Crossref PubMed Scopus (44) Google Scholar). We found that glutaraldehyde significantly increased FRET (Fig. 2C).As a control, a donor-only sample was also treated with glutaraldehyde. Glutaraldehyde caused changes in EDANS fluorescence. Using the same two-state model as above to fit a fraction donor-only and a fraction participating in FRET, we found that the fraction participating in FRET increased from 43% (±5%) to 74% (±1%) with glutaraldehyde treatment (Fig. 2D). The value predicted if all myosin molecules are participating in FRET is 75%, based on the 1:3 donor to acceptor ratio. Thus, we conclude that, when treated with glutaraldehyde, nearly all the myosin molecules are in the IHM.Increasing ionic strength, seen to decrease SRX, did not change the closed-state populationTo test whether the SRX and the IHM are identical, the same myosin was tested under conditions seen to increase the SRX. The ranges of ionic strengths (0–100 mM KCl) tested were previously reported to change the SRX population when measured by stopped-flow mant-ATP turnover kinetics (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). If the SRX and the IHM are identical, then FRET should increase. However, FRET in the samples did not change significantly, remaining at baseline (Fig. 3).Figure 3Increasing ionic strength, seen to decrease super-relaxed state, did not change the closed population. FRET efficiency (E as calculated from amplitude-weighted average lifetimes τ of D and DA waveforms) in range of 0 to 100 mM KCl, (n = 3 per condition, slope found to be not significantly different from zero). FRET, fluorescence resonance energy transfer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mavacamten, a stabilizer of the SRX, increases the closed-state population less than predictedMavacamten is a phase III drug used to treat hypertrophic cardiomyopathy. It has been shown to significantly decrease myosin activity, and electron microscopy has shown that cardiac myosin treated with the cross-linking version of mavacamten was entirely in the IHM state (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). Given this evidence, we predicted that a saturating dose of mavacamten should increase the FRET efficiency from 43% to 74%, if the IHM is fully populated, as predicted by the EM studies.Mavacamten was added to our FRET-labeled sample, and FRET did indeed increase, but only by about 3% (Fig. 4A), an order of magnitude less than predicted. Increasing mavacamten resulted in a hyperbolic response with a Bmax of 4.3% and Kd of 27 μM. The 3% FRET change fits to a model with a 4% increase in the population participating in FRET. The labeled myosin gave the same robust response to mavacamten (33% increase in the population participating in the SRX) as unlabeled myosin in the fluorescent nucleotide turnover kinetics studies (Fig. 4B). When glutaraldehyde was added to samples treated with mavacamten, the population participating in FRET was not significantly different from samples treated with glutaraldehyde alone. Thus, consistent with previous findings, mavacamten increases the IHM population while also increasing the SRX population, but there is an order of magnitude greater effect on the SRX population.Figure 4Mavacamten causes a small increase in FRET but a much larger increase in super-relaxed state population in nucleotide turnover. A, Mavacamten dosage–response curve of FRET of 5 μM labeled heavy meromyosin (n = 3 per condition). B, Single nucleotide turnover of 0.2 μM labeled myosin with 10 μM mavacamten or 1% dimethyl sulfoxide (DMSO) mixed with 4 μM mant-ATP and chased with 2 mM ATP (n = 3 per condition). FRET, fluorescence resonance energy transfer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionMultiple forms of myosin, including mammalian cardiac myosin, appear to have a conserved mechanism of autoinhibition (3Lee K.H. Sulbaran G. Yang S. Mun J.Y. Alamo L. Pinto A. Sato O. Ikebe M. Liu X. Korn E.D. Sarsoza F. Bernstein S.I. Padron R. Craig R. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E1991-E2000Crossref PubMed Scopus (44) Google Scholar). Here we have directly observed the IHM of beta-cardiac myosin, using TR-FRET with randomly distributed donor- and acceptor-labeled RLC. We found that 43% (±5%) of the myosin molecules are in the IHM structural state (Fig. 2D). The SRX biochemical state, measured by fluorescent nucleotide turnover, has been found to vary from 40% to 70% in different beta-cardiac myosin of various species (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar, 17Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human beta-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (125) Google Scholar, 18Toepfer C.N. Garfinkel A.C. Venturini G. Wakimoto H. Repetti G. Alamo L. Sharma A. Agarwal R. Ewoldt J.F. Cloonan P. Letendre J. Lun M. Olivotto I. Colan S. Ashley E. et al.Myosin sequestration regulates sarcomere function, cardiomyocyte energetics, and metabolism, informing the pathogenesis of hypertrophic cardiomyopathy.Circulation. 2020; 141: 828-842Crossref PubMed Scopus (87) Google Scholar). We confirm that 53% (±9%) of the myosin was kinetically SRX in the stopped-flow mant-ATP chase experiments, which is comparable with the 43% of the structurally IHM myosin in the TR-FRET experiments. However, when we tested increasing ionic strength, seen to decrease the SRX population (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar), FRET levels did not decrease. These results suggest that the structurally defined IHM is sufficient but not necessary for the low ATPase activity of the SRX.The predicted distance in most reported models of the interacting heads motif of β-cardiac myosin, which are based on cryo-EM images of tarantula thick filaments (4Alamo L. Pinto A. Sulbaran G. Mavarez J. Padron R. Lessons from a tarantula: New insights into myosin interacting-heads motif evolution and its implications on disease.Biophys. Rev. 2018; 10: 1465-1477Crossref PubMed Scopus (21) Google Scholar, 13Nag S. Trivedi D.V. Sarkar S.S. Adhikari A.S. Sunitha M.S. Sutton S. Ruppel K.M. Spudich J.A. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Nat. Struct. Mol. Biol. 2017; 24: 525-533Crossref PubMed Scopus (94) Google Scholar, 19Robert-Paganin J. Auguin D. Houdusse A. Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition.Nat. Commun. 2018; 9: 4019Crossref PubMed Scopus (46) Google Scholar), is around 5.0 nm, which is longer than the center distance seen by TR-FRET. In those cryo-EM studies, the region of lowest resolution is that of the RLC, suggesting that this area contains more disorder than the rest of the structure, so this discrepancy is not surprising (19Robert-Paganin J. Auguin D. Houdusse A. Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition.Nat. Commun. 2018; 9: 4019Crossref PubMed Scopus (46) Google Scholar). Other factors, such as environmental effects on the fluorescent molecules, may also contribute to this discrepancy.We were unable to observe the open conformation of myosin with this FRET system, indicating that most of the heads on these myosin molecules spend most of their time at least 4 nm apart, as predicted by most models (13Nag S. Trivedi D.V. Sarkar S.S. Adhikari A.S. Sunitha M.S. Sutton S. Ruppel K.M. Spudich J.A. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Nat. Struct. Mol. Biol. 2017; 24: 525-533Crossref PubMed Scopus (94) Google Scholar).We tested whether the shorter distance corresponds to the IHM by treating myosin with glutaraldehyde, a cross-linker used to trap the IHM structure in myosin dimers for electron microscopy (3Lee K.H. Sulbaran G. Yang S. Mun J.Y. Alamo L. Pinto A. Sato O. Ikebe M. Liu X. Korn E.D. Sarsoza F. Bernstein S.I. Padron R. Craig R. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E1991-E2000Crossref PubMed Scopus (44) Google Scholar). We found that glutaraldehyde greatly increased FRET between donor- and acceptor-labeled RLC, confirming the IHM assignment.In this study, we use HMM, a cleaved form of β-cardiac myosin that does not include the whole section of the tail region that is hypothesized to stabilize the IHM. In future, it will be important to use labeled RLC to measure the IHM in full-length myosin, as well as native thick filaments, which would also include accessory proteins that could stabilize IHM.Given the effectiveness of mavacamten to inhibit beta-cardiac myosin contractility (20Kawas R.F. Anderson R.L. Ingle S.R.B. Song Y. Sran A.S. Rodriguez H.M. A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle.J. Biol. Chem. 2017; 292: 16571-16577Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and help patients with hypertrophic cardiomyopathy (21Olivotto I. Oreziak A. Barriales-Villa R. Abraham T.P. Masri A. Garcia-Pavia P. Saberi S. Lakdawala N.K. Wheeler M.T. Owens A. Kubanek M. Wojakowski W. Jensen M.K. Gimeno-Blanes J. Afshar K. et al.Mavacamten for treatment of symptomatic obstructive hypertrophic cardiomyopathy (EXPLORER-HCM): A randomised, double-blind, placebo-controlled, phase 3 trial.Lancet. 2020; 396: 759-769Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), we hypothesized that mavacamten protects the heart through sequestering of myosin heads in the inactive SRX biochemical state, which is outside of the mechanochemical cycle. Our laboratory and others have found that mavacamten potently increases the mole fraction of the SRX population (measured by nucleotide exchange) of β-cardiac myosin (16Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E7486-E7494Crossref PubMed Google Scholar, 17Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human beta-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (125) Google Scholar). However, we observed that mavacamten did not greatly increase the mole ratio of the IHM state (measured structurally by FRET). Since our study detects the structural state of IHM under conditions (no freezing, no fixing) similar to those used previously to detect SRX, these results call into question the unconfirmed hypothesis that there is a one-to-one correspondence between the IHM structural state and the SRX biochemical state. Our results point to multiple structural states leading to SRX-like kinetics, one being the IHM and the other indistinguishable (by FRET) from the open conformation.Based on our current results, we hypothesize that mavacamten does not fully trap cardiac myosin in the IHM. This may explain why it successfully treats hypertrophic cardiomyopathy without completely stopping heart contractions. Studies in cardiac S1, a cleaved single-headed version of myosin, showed that mavacamten primarily inhibits myosin by slowing the phosphate release and ADP release steps in the mechanochemical cycle, without head–head interaction (20Kawas R.F. Anderson R.L. Ingle S.R.B. Song Y. Sran A.S. Rodriguez H.M. A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle.J. Biol. Chem. 2017; 292: 16571-16577Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The slowing of these rates may also slow the single-turnover kinetics, leading to SRX-like rates. Our results show that the IHM-like structure leads to SRX-like kinetics but that the population of myosin in SRX can greatly exceed that in the IHM. Thus, there is a second (structurally distinct) population of SRX myosin having kinetics thought to be cardioprotective, but that does not require static sequestration via the IHM structure.Experimental proceduresProtein preparationsβ-Cardiac myosin was purified from bovine hearts obtain
Referência(s)