A study of tropomyosin's role in cardiac function and disease using thin-filament reconstituted myocardium
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Tropomyosin (Tm) is the key regulatory com- ponent of the thin-filament and plays a central role in the cardiac muscle's cooperative activation mechanism. Many mutations of cardiac Tm are related to hypertrophic car- diomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC). Using the thin- filament extraction/reconstitution technique, we are able to incorporate various Tm mutants and protein isoforms into a muscle fiber environment to study their roles in Ca 2? regulation, cross-bridge kinetics, and force generation. The thin-filament reconstitution technique poses several advantages compared to other in vitro and in vivo methods: (1) Tm mutants and isoforms are placed into the real muscle fiber environment to exhibit their effect on a level much higher than simple protein complexes; (2) only the primary and immediate effects of Tm mutants are studied in the thin-filament reconstituted myocardium; (3) lethal mutants of Tm can be studied without causing a problem; and (4) inexpensive. In transgenic models, various sec- ondary effects (myocyte disarray, ECM fibrosis, altered protein phosphorylation levels, etc.) also affect the per- formance of the myocardium, making it very difficult to isolate the primary effect of the mutation. Our studies on Tm have demonstrated that: (1) Tm positively enhances the hydrophobic interaction between actin and myosin in the ''closed state'', which in turn enhances the isometric ten- sion; (2) Tm's seven periodical repeats carry distinct functions, with the 3rd period being essential for the ten- sion enhancement; (3) Tm mutants lead to HCM by impairing the relaxation on one hand, and lead to DCM by over inhibition of the AM interaction on the other hand. Ca 2? sensitivity is affected by inorganic phosphate, ionic strength, and phosphorylation of constituent proteins; hence it may not be the primary cause of the pathogenesis. Here, we review our current knowledge regarding Tm's effect on the actomyosin interaction and the early molec- ular pathogenesis of Tm mutation related to HCM, DCM, and LVNC.Keywords:
Tropomyosin
Cardiac muscle
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Pathogenesis of most myopathies including inherited hypertrophic (HCM) and dilated (DCM) cardiomyopathies is based on modification of structural state of contractile proteins induced by point mutations, such as mutations in alpha-tropomyosin (TM). To understand the mechanism of abnormal function of contractile system of muscle fiber due to Glu180Gly, Asp175 or Glu40Lys, Glu54Lys mutations in alpha-TM associated with HCM or DCM, we specifically labeled alpha-TM by fluorescence probe 5-IAF after Cys-190 and examined the position and mobility of the IAF-TM in the ATP hydrolysis cycle using polarized fluorescence technique. Analysis of the data suggested that the point mutations in alpha-TM associated with hypertrophic or dilated cardiomyopathy caused abnormal changes in the affinity ofTM to actin and in the position of this protein on the thin filaments in the ATPase cycle. Mutations in alpha-TM associated with HCM caused a shift of TM strands to the center of the thin filament and increased a range of tropomyosin motion and affinity of this protein to actin in the ATPase cycle. In contrast, mutations in alpha-TM associated with DCM shifted the protein to the periphery of the thin filament, reduced the amplitude of the TM movement and its affinity for actin. It is proposed that anomalous behavior of TM on the thin filaments in ATPase cycle may provoke the dysfunction of the cardiac muscle in patients with HCM and DCM.
Tropomyosin
Dilated Cardiomyopathy
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Mammalian cardiac and skeletal muscle express unique isoforms of the thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), and the significance of these different isoforms in thin filament regulation has not been clearly identified. Both in vitro and skinned cellular studies investigating the mechanism of thin filament regulation in striated muscle have often used heterogeneous mixtures of Tn, Tm and myosin isoforms, and variability in reported results might be explained by different combinations of these proteins. Here we used in vitro motility and force (microneedle) assays to investigate the influence of cardiac versus skeletal Tn and Tm isoforms on actin–heavy meromyosin (HMM) mechanics. When interacting with skeletal HMM, thin filaments reconstituted with cardiac Tn/Tm or skeletal Tn/Tm exhibited similar speed–calcium relationships and significantly increased maximum speed and force per filament length ( F / l ) at pCa 5 ( versus unregulated actin filaments). However, augmentation of F / l was greater with skeletal regulatory proteins. Reconstitution of thin filaments with the heterogeneous combination of skeletal Tn and cardiac Tm decreased sliding speeds at all [Ca 2+ ] relative to thin filaments with skeletal Tn/Tm. Finally, for filaments reconstituted with any heterogeneous mix of Tn and Tm isoforms, force was not potentiated over that of unregulated actin filaments. Combined the results suggest (1) that cardiac regulatory proteins limit the allosteric enhancement of force, and (2) that Tn and Tm isoform homogeneity is important when studying Ca 2+ regulation of crossbridge binding and kinetics as well as mechanistic differences between cardiac and skeletal muscle.
Tropomyosin
Heavy meromyosin
CrossBridge
Cardiac muscle
Meromyosin
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Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin–tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin–tropomyosin binding and weaker tropomyosin–actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin–thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM. Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin–tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin–tropomyosin binding and weaker tropomyosin–actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin–thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM. Cardiac muscle contraction, generated by the ATP-dependent attachment and detachment of myosin and actin, is regulated by a variety of proteins along the thick and thin filaments of the sarcomere. Sarcomeric protein mutations have been shown to have profound effects on cross-bridge cycling and often interfere with the proper regulation of contraction (1Chang A.N. Potter J.D. Sarcomeric protein mutations in dilated cardiomyopathy.Heart Fail. Rev. 2005; 10 (16416045): 225-23510.1007/s10741-005-5252-6Crossref PubMed Scopus (104) Google Scholar). Inherited, autosomal dominant mutations are related to more than 80% of clinical cases of dilated cardiomyopathy (DCM); these mutations frequently occur in sarcomeric protein genes including the thin filament proteins actin, all three of the troponin subunits, and tropomyosin (2Redwood C. Robinson P. α-Tropomyosin mutations in inherited cardiomyopathies.J. Muscle Res. Cell Motil. 2013; 34 (24005378): 285-29410.1007/s10974-013-9358-5Crossref PubMed Scopus (55) Google Scholar). Understanding the effects of these mutations on protein–protein interaction is needed to inform future therapeutic design and personalized drugs of choice (3Tardiff J.C. Carrier L. Bers D.M. Poggesi C. Ferrantini C. Coppini R. Maier L.S. Ashrafian H. Huke S. van der Velden J. Targets for therapy in sarcomeric cardiomyopathies.Cardiovasc. Res. 2015; 105 (25634554): 457-47010.1093/cvr/cvv023Crossref PubMed Scopus (87) Google Scholar). Tropomyosin molecules twist together as a parallel dimer that forms an α-helical coiled-coil. The tightly wound nature of tropomyosin provides little protection from local mutation-induced structural changes having long-range effects (4Farman G.P. Rynkiewicz M.J. Orzechowski M. Lehman W. Moore J.R. HCM and DCM cardiomyopathy-linked α-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments.Arch. Biochem. Biophys. 2018; 647 (29626422): 84-9210.1016/j.abb.2018.04.002Crossref PubMed Scopus (10) Google Scholar, 5Sundar S. Rynkiewicz M.J. Ghosh A. Lehman W. Moore J.R. Cardiomyopathy mutation alters end-to-end junction of tropomyosin and reduces calcium sensitivity.Biophys. J. 2020; 118 (31882250): 303-31210.1016/j.bpj.2019.11.3396Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar) that can act a hundred or more angstroms away from the site of the mutation (4Farman G.P. Rynkiewicz M.J. Orzechowski M. Lehman W. Moore J.R. HCM and DCM cardiomyopathy-linked α-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments.Arch. Biochem. Biophys. 2018; 647 (29626422): 84-9210.1016/j.abb.2018.04.002Crossref PubMed Scopus (10) Google Scholar, 6Li X.E. Suphamungmee W. Janco M. Geeves M.A. Marston S.B. Fischer S. Lehman W. The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy.Biochem. Biophys. Res. Commun. 2012; 424 (22789852): 493-49610.1016/j.bbrc.2012.06.141Crossref PubMed Scopus (41) Google Scholar). Each tropomyosin dimer binds seven consecutive actin subunits and, when bound end-to-end with adjacent tropomyosin dimers, creates a continuous cable that wraps around the actin filament. The overlap of ∼11 residues of each terminus of neighboring tropomyosin dimers form a four-helix bundle (7Greenfield N.J. Huang Y.J. Swapna G.V. Bhattacharya A. Rapp B. Singh A. Montelione G.T. Hitchcock-DeGregori S.E. Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation.J. Mol. Biol. 2006; 364 (16999976): 80-9610.1016/j.jmb.2006.08.033Crossref PubMed Scopus (109) Google Scholar). The N terminus of the troponin subunit T forms a complex with this head-to-tail four-helix bundle of tropomyosin (8Pavadai E. Rynkiewicz M.J. Ghosh A. Lehman W. Docking troponin T onto the tropomyosin overlapping domain of thin filaments.Biophys. J. 2020; 118 (31864661): 325-33610.1016/j.bpj.2019.11.3393Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 9Yamada Y. Namba K. Fujii T. Cardiac muscle thin filament structures reveal calcium regulatory mechanism.Nat. Commun. 2020; 11 (31919429): 15310.1038/s41467-019-14008-1Crossref PubMed Scopus (40) Google Scholar), offering additional stabilization (10Pinto J.R. Gomes A.V. Jones M.A. Liang J. Nguyen S. Miller T. Parvatiyar M.S. Potter J.D. The functional properties of human slow skeletal troponin T isoforms in cardiac muscle regulation.J. Biol. Chem. 2012; 287 (22977240): 37362-3737010.1074/jbc.M112.364927Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 11Sousa D. Cammarato A. Jang K. Graceffa P. Tobacman L.S. Li X.E. Lehman W. Electron microscopy and persistence length analysis of semi-rigid smooth muscle tropomyosin strands.Biophys. J. 2010; 99 (20682264): 862-86810.1016/j.bpj.2010.05.004Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), which slows myosin–ATPase activity (12Tobacman L.S. Nihli M. Butters C. Heller M. Hatch V. Craig R. Lehman W. Homsher E. The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity.J. Biol. Chem. 2002; 277 (12011043): 27636-2764210.1074/jbc.M201768200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) when assessed in vitro. The C terminus of troponin T and the troponin complex subunits I and C bind both to the central portion of the tropomyosin dimer and to actin (9Yamada Y. Namba K. Fujii T. Cardiac muscle thin filament structures reveal calcium regulatory mechanism.Nat. Commun. 2020; 11 (31919429): 15310.1038/s41467-019-14008-1Crossref PubMed Scopus (40) Google Scholar, 13Jin J.-P. Chong S.M. Localization of the two tropomyosin-binding sites of troponin T.Arch. Biochem. Biophys. 2010; 500 (20529660): 144-15010.1016/j.abb.2010.06.001Crossref PubMed Scopus (66) Google Scholar, 14Palm T. Greenfield N.J. Hitchcock-DeGregori S.E. Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes.Biophys. J. 2003; 84 (12719247): 3181-318910.1016/S0006-3495(03)70042-3Abstract Full Text Full Text PDF PubMed Google Scholar). Each tropomyosin dimer interacts weakly with actin primarily via electrostatic interactions (15Li X.E. Tobacman L.S. Mun J.Y. Craig R. Fischer S. Lehman W. Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry.Biophys. J. 2011; 100 (21320445): 1005-101310.1016/j.bpj.2010.12.3697Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 16Lorenz M. Poole K.J. Popp D. Rosenbaum G. Holmes K.C. An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin–tropomyosin gels.J. Mol. Biol. 1995; 246 (7853391): 108-11910.1006/jmbi.1994.0070Crossref PubMed Google Scholar, 17Rynkiewicz M.J. Prum T. Hollenberg S. Kiani F.A. Fagnant P.M. Marston S.B. Trybus K.M. Fischer S. Moore J.R. Lehman W. Tropomyosin must interact weakly with actin to effectively regulate thin filament function.Biophys. J. 2017; 113 (29211998): 2444-245110.1016/j.bpj.2017.10.004Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar); however, the observed high affinity of tropomyosin for actin results from the end-to-end bonds between adjacent tropomyosin dimers, which stabilize the tropomyosin cable along the long-pitch helix of actin, preventing tropomyosin detachment. This binding arrangement allows for tropomyosin to saturate the thin filament while also allowing movement between three regulatory states (18Lehman W. Switching muscles on and off in steps: the McKillop–Geeves three-state model of muscle regulation.Biophys. J. 2017; 112 (28552313): 2459-246610.1016/j.bpj.2017.04.053Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 19McKillop D.F. Geeves M.A. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament.Biophys. J. 1993; 65 (8218897): 693-70110.1016/S0006-3495(93)81110-XAbstract Full Text PDF PubMed Google Scholar). In the absence of calcium, the regulatory complex consisting of troponin and tropomyosin blocks the myosin-binding sites along actin, preventing the myosin-actin strong-binding state. At higher calcium levels, the troponin C subunit binds calcium, resulting in a cascade of conformational changes that lead to a shift in tropomyosin from the "blocked" state, favoring "closed" and "open" states, each of which has a structural correlate: the thin filament "B-state," "C-state," and "M-state" positions. This process allows myosin heads to interact with actin and undergo cross-bridge cycling to generate force (11Sousa D. Cammarato A. Jang K. Graceffa P. Tobacman L.S. Li X.E. Lehman W. Electron microscopy and persistence length analysis of semi-rigid smooth muscle tropomyosin strands.Biophys. J. 2010; 99 (20682264): 862-86810.1016/j.bpj.2010.05.004Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 15Li X.E. Tobacman L.S. Mun J.Y. Craig R. Fischer S. Lehman W. Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry.Biophys. J. 2011; 100 (21320445): 1005-101310.1016/j.bpj.2010.12.3697Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 18Lehman W. Switching muscles on and off in steps: the McKillop–Geeves three-state model of muscle regulation.Biophys. J. 2017; 112 (28552313): 2459-246610.1016/j.bpj.2017.04.053Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Strong tropomyosin head–tail bonds allow the azimuthal shift of tropomyosin to be propagated between adjacent tropomyosin dimers along the thin filament, thus playing a role in cooperative activation of actin–myosin interactions. Therefore, the stiffness of the tropomyosin dimer, the affinity of tropomyosin for actin and troponin, and the strength of the tropomyosin–tropomyosin bond all will influence the transition between regulatory states and ultimately control muscle contraction. Mutations in the tropomyosin–tropomyosin overlap region can cause devastating and progressive dysfunction of the heart (20Hershberger R.E. Norton N. Morales A. Li D. Siegfried J.D. Gonzalez-Quintana J. Coding sequence rare variants identified in MYBPC3, MYH6, TPM1, TNNC1 and TNNI3 from 312 patients with familial or idiopathic dilated cardiomyopathy.Circ. Cardiovasc. Genet. 2010; 3 (20215591): 155-16110.1161/CIRCGENETICS.109.912345Crossref PubMed Scopus (155) Google Scholar, 21Lakdawala N.K. Funke B.H. Baxter S. Cirino A.L. Roberts A.E. Judge D.P. Johnson N. Mendelsohn N.J. Morel C. Care M. Chung W.K. Jones C. Psychogios A. Duffy E. Rehm H.L. et al.Genetic testing for dilated cardiomyopathy in clinical practice.J. Card. Fail. 2012; 18 (22464770): 296-30310.1016/j.cardfail.2012.01.013Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 22Van Driest S.L. Ellsworth E.G. Ommen S.R. Will M.L. Tajik A.J. Gersh B.J. Ackerman M.J. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy.Circulation. 2003; 108 (12860912): 445-45110.1161/01.CIR.0000080896.52003.DFCrossref PubMed Scopus (178) Google Scholar). For example the methionine at position 8 is part of the four-helix bundle's hydrophobic core in the tropomyosin overlap complex (7Greenfield N.J. Huang Y.J. Swapna G.V. Bhattacharya A. Rapp B. Singh A. Montelione G.T. Hitchcock-DeGregori S.E. Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation.J. Mol. Biol. 2006; 364 (16999976): 80-9610.1016/j.jmb.2006.08.033Crossref PubMed Scopus (109) Google Scholar, 23Rao J.N. Rivera-Santiago R. Li X.E. Lehman W. Dominguez R. Structural analysis of smooth muscle tropomyosin α and β isoforms.J. Biol. Chem. 2012; 287 (22119916): 3165-317410.1074/jbc.M111.307330Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and when mutated is connected to DCM or to nemaline myopathy (21Lakdawala N.K. Funke B.H. Baxter S. Cirino A.L. Roberts A.E. Judge D.P. Johnson N. Mendelsohn N.J. Morel C. Care M. Chung W.K. Jones C. Psychogios A. Duffy E. Rehm H.L. et al.Genetic testing for dilated cardiomyopathy in clinical practice.J. Card. Fail. 2012; 18 (22464770): 296-30310.1016/j.cardfail.2012.01.013Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 24Ilkovski B. Mokbel N. Lewis R.A. Walker K. Nowak K.J. Domazetovska A. Laing N.G. Fowler V.M. North K.N. Cooper S.T. Disease severity and thin filament regulation in M9R TPM3 nemaline myopathy.J. Neuropathol. Exp. Neurol. 2008; 67 (18716557): 867-87710.1097/NEN.0b013e318183a44fCrossref PubMed Scopus (19) Google Scholar). Here, the mutation to a positively charged arginine in α-tropomyosin is expected to disrupt the four-helix bundle structure and perturb biochemical and functional properties. To test the effects of the M8R mutation in tropomyosin on actomyosin contractile activity, we expressed tropomyosin containing the Met-to-Arg substitution in Escherichia coli. Modeling the junctional tropomyosin–tropomyosin interactions following molecular dynamics (MD) simulations showed that the solvent-accessible surface area of the four-helix bundle was much greater in the M8R tropomyosin, consistent with the observed alterations in tropomyosin structure and reduced tropomyosin–tropomyosin end-to-end bond strength. Furthermore, MD simulations of mutant tropomyosin on actin indicate structural alterations that correspond to disrupted regulatory transitions of tropomyosin on the actin filament. Measurements of co-sedimentation in the presence and absence of a troponin T N-terminal fragment suggested that the structural change in the four-helix bundle diminished troponin T's ability to bind to actin–tropomyosin. In addition, the in vitro motility assay was used to assess the ability of the mutant tropomyosin to properly confer calcium regulation of actin propulsion by myosin and shows that the cooperativity and calcium sensitivity of the troponin–tropomyosin regulatory complex were reduced. Taken together, these data demonstrate that both local and long-range perturbations in tropomyosin structure underlie DCM mutation-induced changes in actin–tropomyosin interactions that contribute to disease development. The N terminus of tropomyosin is a critical determinant of tropomyosin's ability to interact with actin and to perform its regulatory functions in striated muscle (14Palm T. Greenfield N.J. Hitchcock-DeGregori S.E. Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes.Biophys. J. 2003; 84 (12719247): 3181-318910.1016/S0006-3495(03)70042-3Abstract Full Text Full Text PDF PubMed Google Scholar). Using CD, we determined that both WT and M8R mutant tropomyosin exhibit the characteristic pattern for an α-helical twist, with a peak at 222 nm that is typically used to quantify the presence of the α-helix (Fig. 1A) (25Bennion B.J. Daggett V. The molecular basis for the chemical denaturation of proteins by urea.Proc. Natl. Acad. Sci. U.S.A. 2003; 100 (12702764): 5142-514710.1073/pnas.0930122100Crossref PubMed Scopus (636) Google Scholar). The three-peaked ellipticity displayed by the M8R tropomyosin indicates that, despite the amino acid substitution, it still forms an α-helical structure. Chemical denaturation, determined by monitoring tropomyosin CD ellipticity at 220 nm when the protein is exposed to increasing concentrations of urea (0–4 m), was used to determine the effects of the M8R mutation on the stability of the mutant's secondary and tertiary structure (25Bennion B.J. Daggett V. The molecular basis for the chemical denaturation of proteins by urea.Proc. Natl. Acad. Sci. U.S.A. 2003; 100 (12702764): 5142-514710.1073/pnas.0930122100Crossref PubMed Scopus (636) Google Scholar) (Fig. 1B). The M8R tropomyosin unfolding following urea treatment demonstrated a decreased overall stability (CUrea-unfold at 1.52 ± 0.09 m versus WT 1.98 ± 0.05 m) and less resistance to chemical denaturation (nHUrea-unfold 0.77 ± 0.15 versus WT 1.23 ± 0.17), suggesting that the M8R tropomyosin dimer was folded less compactly compared with WT. Because tropomyosin dimers form intermittent bonds to one another in solution, it is not apparent from these data alone whether the four-helix bundle in particular is affected or whether it is the N terminus alone that has been changed. Previous computational work by our group on the head-to-tail bond between two tropomyosin dimers showed a compact N-terminal coiled-coil penetrating slightly spread coils of the C terminus to form a four-helix bundle, which is stabilized by hydrophobic interactions (26Li X.E. Orzechowski M. Lehman W. Fischer S. Structure and flexibility of the tropomyosin overlap junction.Biochem. Biophys. Res. Commun. 2014; 446 (24607906): 304-30810.1016/j.bbrc.2014.02.097Crossref PubMed Scopus (30) Google Scholar). Substitution of a charged arginine side chain at the methionine 8 position may disrupt the hydrophobic core of the overlap and expand the overlap domain, thus altering its structural and mechanical properties. We therefore examined the local effects of the M8R mutation on the formation and stability of the end-to-end bond four-helix bundle via MD simulation. Met8 of WT tropomyosin projects into the core of the tropomyosin end-to-end bundle; however, the four-helix bundle containing the M8R substitution transitioned to three helixes with a strand pushed out of the hydrophobic core, allowing for the arginine to project outward into solution (Fig. 2), resulting in an increased bending angle across the overlap (12 ± 2.7 versus WT 9.3 ± 4.8). This altered structure decreased the helix's buried solvent-accessible surface area (M8R in isolation 2212 ± 73 Å versus WT 2374 ± 65 Å2), consistent with the observed increased susceptibility to chemical denaturants (Fig. 1). To perform its regulatory function, each tropomyosin molecule must interact weakly with actin to assure the ability to shift between regulatory positions. With such low-affinity interactions with actin, saturation of actin with tropomyosin relies on the tropomyosin–tropomyosin end-to-end linkage where tropomyosin dimers bind to adjacent dimers forming a chain along the thin filament. Given the M8R-induced structural disruptions observed in the molecular dynamics simulations, we therefore determined the effect of the M8R mutation on the binding of tropomyosin to actin. Consistent with previous studies, M8R tropomyosin exhibits a dramatically lowered actin affinity reaching the limit of sensitivity for the assay (27Moraczewska J. Greenfield N.J. Liu Y. Hitchcock-DeGregori S.E. Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.Biophys. J. 2000; 79 (11106625): 3217-322510.1016/S0006-3495(00)76554-4Abstract Full Text Full Text PDF PubMed Google Scholar) (Fig. 3, A–C). By incubating 0.55 μm M8R tropomyosin with actin in the presence of increasing concentrations of S1 (up to 2 μm), we see that the M8R tropomyosin approaches the maximum, fully saturated WT tropomyosin/actin-binding ratio (Fig. 3, D and E). Therefore, consistent with previous studies (27Moraczewska J. Greenfield N.J. Liu Y. Hitchcock-DeGregori S.E. Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.Biophys. J. 2000; 79 (11106625): 3217-322510.1016/S0006-3495(00)76554-4Abstract Full Text Full Text PDF PubMed Google Scholar), M8R tropomyosin was capable of binding actin in the presence of myosin subunit S1, which is known to increase tropomyosin–actin affinity and favor the M-state position of tropomyosin on actin (27Moraczewska J. Greenfield N.J. Liu Y. Hitchcock-DeGregori S.E. Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.Biophys. J. 2000; 79 (11106625): 3217-322510.1016/S0006-3495(00)76554-4Abstract Full Text Full Text PDF PubMed Google Scholar). To examine the impact of the M8R mutation on the structure of, and interactions between, tropomyosin and actin, we performed molecular dynamics simulations of WT and mutant tropomyosin on actin. To study mutations in the tropomyosin end-to-end overlap, we exploited our recently developed model of actin–tropomyosin (8Pavadai E. Rynkiewicz M.J. Ghosh A. Lehman W. Docking troponin T onto the tropomyosin overlapping domain of thin filaments.Biophys. J. 2020; 118 (31864661): 325-33610.1016/j.bpj.2019.11.3393Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) derived from cryo-EM of the thin filament (9Yamada Y. Namba K. Fujii T. Cardiac muscle thin filament structures reveal calcium regulatory mechanism.Nat. Commun. 2020; 11 (31919429): 15310.1038/s41467-019-14008-1Crossref PubMed Scopus (40) Google Scholar) that utilizes periodic boundary conditions to create an essentially infinite filament, as described previously (8Pavadai E. Rynkiewicz M.J. Ghosh A. Lehman W. Docking troponin T onto the tropomyosin overlapping domain of thin filaments.Biophys. J. 2020; 118 (31864661): 325-33610.1016/j.bpj.2019.11.3393Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Comparison of the WT and M8R simulations show a similar result to the isolated overlap simulations, where the mutant arginine side chain moves from the inside of the four-helix bundle to a solvent-exposed position, with concurrent displacement of the helices in the bundle; a comparison of the isolated overlap and tropomyosin-on-actin structures shows relatively little change in the M8R tropomyosin helices, with the exception of the helix-contacting actin (Fig. 4). However, the geometry of the overlap is relatively intact, because there is little change between WT and M8R in the curvature (7.1 ± 2.0° versus 7.9 ± 3.1°, respectively), the junction twist angle (84.7 ± 1.9° versus 85.4 ± 2.1, respectively), or the buried solvent-exposed surface area in the overlap domain (2173 ± 61 Å2 versus 2135 ± 84 Å2, respectively). This result could be a consequence of restricting the tropomyosin cable to the actin filament, which may limit the accessible conformations in this simulation. However, further inspection reveals some critical differences between the WT and M8R structures. In the WT, the dynamic persistence length of the overlap domain, which is associated with cooperativity of the thin filament, increases from 341 nm in isolation to 1969 nm when part of an infinite cable, an ∼6-fold increase in stiffness consistent with topologically constraining the overlap to the filament. However, the overlap dynamic persistence length of the M8R mutant is relatively unchanged when comparing the simulation in isolation (1080 nm) and in a cable (819 nm). Notably, the overlap dynamic persistence length is reduced in the cable by a factor of 2, suggesting that the mutation has created a less-stiff overlap. The local distortions observed in the simulations are also predicted to alter tropomyosin's regulatory properties. The distortions to the overlap domain affect the troponin T–binding site. Although Met8 does not make direct contacts to troponin T in the recently published model (8Pavadai E. Rynkiewicz M.J. Ghosh A. Lehman W. Docking troponin T onto the tropomyosin overlapping domain of thin filaments.Biophys. J. 2020; 118 (31864661): 325-33610.1016/j.bpj.2019.11.3393Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), flanking residues Lys6, Lys7, Gln9, and Met10 interact with the troponin T N terminus. Thus, alterations to this region of tropomyosin are predicted to alter troponin T binding and regulatory function. Second, comparison of representative frames from the simulations on actin show that the mutation has resulted in a significant shift away from its starting, high-calcium C-state position toward the low-calcium position (Fig. 5A). The mutation-induced alterations of the overlap structure appear to change the twist of the overlap domain to a more B-state–like structure (Fig. 5C), and this twist is then propagated throughout the tropomyosin cable, stopping at the middle part of tropomyosin. These results suggest that the M8R cable adopts a conformation that would block myosin binding more effectively than the WT cable. Thus, the model suggests that the mutant filament would require a greater calcium concentration to achieve full activity. The N-terminal segment of troponin T binds across the end-to-end junction of adjacent tropomyosin molecules on the actin filament (diagram of Fig. 5D) and is known to depress the speed of tropomyosin-bound actin filaments at low myosin concentrations (12Tobacman L.S. Nihli M. Butters C. Heller M. Hatch V. Craig R. Lehman W. Homsher E. The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity.J. Biol. Chem. 2002; 277 (12011043): 27636-2764210.1074/jbc.M201768200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We determined the speed of tropomyosin-decorated actin filaments in the presence of the troponin T fragment to determine whether structural and biochemical alterations caused by the M8R tropomyosin mutation affect troponin T function in tropomyosin–actin filaments. M8R-tropomyosin–decorated filaments showed no alteration in filament speed, although the troponin T-fragment did reduce WT tropomyosin speed, suggesting either that troponin T fragment binding to M8R tropomyosin is compromised or that the effect of the troponin T N terminus on filament sliding is lost in the presence of the M8R mutation (Fig. 6A). To further elucidate why the troponin T N terminus fragment failed to affect the M8R-tropomyosin–regulated actin in the in vitro motility assay, the ability of troponin T1 to actin filaments with bound mutant M8R- and WT-tropomyosin was determined via co-sedimentation. At saturating concentration for troponin T1 fragment binding to WT tropomyosin (5Sundar S. Rynkiewicz M.J. Ghosh A. Lehman W. Moore J.R. Cardiomyopathy mutation
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Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca2+ binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament. Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca2+ binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament. familial hypertrophic cardiomyopathy troponin Familial hypertrophic cardiomyopathy (FHC)1 is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus (1Geisterfer-Lowrance A.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Abstract Full Text PDF PubMed Scopus (1039) Google Scholar, 2Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. Seidman C.E. N. Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (774) Google Scholar, 3Charron P. Dubourg O. Desnos M. Bennaceur M. Carrier L. Camproux A.C. Isnard R. Hagege A. Langlard J.M. Bonne G. Richard P. Hainque B. Bouhour J.B. Schwartz K. Komajda M. Circulation. 1998; 97: 2230-2236Crossref PubMed Scopus (211) Google Scholar, 4Kimura A. Harada H. Park J.-E. Nishi H. Satoh M. Takahashi M. Hiroi S. Sasaoka T. Ohbuchi N. Nakamura T. Koyanagi T. Hwang T.-H. Choo J.-A. Chung K.-S. Hasegawa A. Nagai R. Okazaki O. Nakamura H. Matsuzaki M. Sakamoto T. Toshima H. Koga Y. Imaizumi T. Sasazuki T. Nat. Genet. 1997; 16: 379-382Crossref PubMed Scopus (468) Google Scholar). Despite the varied functions of these many proteins, the clinical and histological manifestations of FHC define a common syndrome involving thickening of one or more parts of the left ventricular wall, myocyte disarray, fibrosis, and a variety of cardiac symptoms including sudden death (reviewed in Ref. 5Spirito P. Seidman C.E. McKenna W.J. Maron B.J. N. Engl. J. Med. 1997; 336: 775-785Crossref PubMed Scopus (875) Google Scholar). Cardiomyopathic mutations have been described for thick filament proteins, as well as for every thin filament component except troponin C (TnC), i.e. for α-tropomyosin and cardiac actin and troponin I and T (TnI, TnT). TnT appears to be the most frequent thin filament target, and mutations in TnT are associated with relatively high mortality despite only modest cardiac hypertrophy (2Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. Seidman C.E. N. Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (774) Google Scholar). Experimentally, TnT mutations produce physiological dysfunction in transgenic animals and in cultured cells and altered function of purified proteins assessed in vitro (reviewed in Ref. 6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, the underlying mechanisms leading to these dysfunction(s) remain poorly understood. Both the thin filament and the thick filament are dynamic interacting protein assemblies. Large structural transitions in myosin produce the cross-bridge stroke that results in muscle contraction (7Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 8Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). Similarly, changes in thin filament structure are critical for Ca2+ regulation of contraction (9Holmes K.C. Biophys. J. 1995; 68: 2s-7sPubMed Google Scholar, 10Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar, 11Squire J.M. Morris E.P. FASEB J. 1998; 12: 761-771Crossref PubMed Scopus (168) Google Scholar). FHC mutations in either filament presumably act by altering filament structure or dynamics, although no direct structural examination of FHC mutants has been reported. However, critical insights into the basis of FHC have come from mapping myosin mutations onto the atomic model of the myosin head (12Rayment I. Holden H.M. Sellers J.R. Fananapazir L. Epstein N.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3864-3868Crossref PubMed Scopus (193) Google Scholar, 13Ruppel K.M. Spudich J.A. Annu. Rev. Cell Dev. Biol. 1996; 12: 543-573Crossref PubMed Scopus (73) Google Scholar). Similarly for the thin filament, mutations can be mapped on to the atomic structures of the components where these are available. However, a full understanding of thin filament mutations has not been possible because of the lack of an atomic model of the thin filament as a whole and because no direct structural studies have been performed. Our recent elucidation of thin filament molecular structure by three-dimensional reconstruction of electron micrographs approaches such a model and has provided essential structural insights into the thin filament regulatory mechanism (14Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 15Korman V.L. Hatch V. Dixon K. Craig R. Lehman W. Tobacman L.S. J. Biol. Chem. 2000; 275: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). These studies show that tropomyosin adopts three distinct positions on actin depending on Ca2+ binding to troponin and myosin binding to actin (10Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar). In the absence of Ca2+, tropomyosin is localized on the periphery of the filament, where it sterically inhibits actin-myosin interaction, thereby causing relaxation (9Holmes K.C. Biophys. J. 1995; 68: 2s-7sPubMed Google Scholar, 16Lehman W. Craig R. Vibert P. Nature. 1994; 368: 65-67Crossref PubMed Scopus (271) Google Scholar). Activation results from a two-step movement of tropomyosin away from the myosin binding site, the first induced by Ca2+, partially switching on the thin filament, and the second by myosin head binding leading to full activation (10Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar). Our structural experiments have also enabled functional changes to be correlated with perturbations of regulatory transitions in thin filament structure. In this paper, we correlate the structural and functional effects of a FHC mutation in TnT to characterize the disease at the molecular level. We examine a 28-residue COOH-terminal truncation of cardiac TnT, similar to protein resulting from a FHC splice site mutation at the beginning of intron 15 (2). Heterozygotes for this mutation experience ∼25% mortality by age 25, similar to the mortality associated with other TnT mutations (2Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. Seidman C.E. N. Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (774) Google Scholar). In transgenic animal models expressing the mutant protein, both systolic and diastolic function are compromised (17Tardiff J.C. Factor S.M. Tompkins B.D. Hewett T.E. Palmer B.M. Moore R.L. Schwartz S. Robbins J. Leinwand L.A. J. Clin. Invest. 1998; 101: 2800-2811Crossref PubMed Scopus (164) Google Scholar). Moreover, in a variety of in vitro experimental systems, thin filaments containing this mutation exhibit impaired regulation of actin-myosin interactions reflected in reduced inhibition of actomyosin ATPase activity in the absence of Ca2+, diminished activation of myosin cycling in the presence of Ca2+ (6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 19Mukherjea P. Tong L. Seidman J.G. Seidman C.E. Hitchcock-DeGregori S.E. Biochemistry. 1999; 38: 13296-13301Crossref PubMed Scopus (23) Google Scholar), and diminished force (18Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 20Watkins H.C. Seidman C.E. Seidman J.G. Feng H.S. Sweeney H.L. J. Clin. Invest. 1996; 98: 2456-2461Crossref PubMed Scopus (112) Google Scholar). Despite the obviously altered control mechanisms, the present report shows that tropomyosin adopts normal positions on the actin filament, both in the presence and in the absence of Ca2+. The origin of the thin filament functional abnormalities is instead shown to be due to weakened binding of troponin to the thin filament to different extents in the three regulatory states, thereby affecting the transitions among these states that control myosin binding and regulate contraction. Development of hypertrophic cardiomyopathy due to this mutation can thus be traced to a defect in the energetics of thin filament conformational switching. Rabbit fast skeletal muscle actin and myosin subfragment 1 were purified to homogeneity as described previously (14Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Cardiac tropomyosin and troponin subunits were purified (14Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) from bovine heart obtained at a local slaughterhouse. Bovine cardiac TnT containing a 28-residue COOH-terminal truncation was expressed in DE3 cells using the pET3d-based expression vector, as described previously (6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), as was wild type recombinant TnT. In humans, the FHC-inducing splice site mutation in cardiac TnT, intron 15 G1A, results in two truncated proteins, one missing 14 COOH-terminal residues, and the other in which the 28 COOH-terminal residues are replaced by seven novel residues. In some experiments (as indicated), TnT was carboxymethylated on Cys39 using [3H]iodoacetic acid (Amersham Pharmacia Biotech). Labeled and unlabeled troponins were reconstituted by combining TnI, TnC, and TnT under denaturing conditions in a 1:1:1 mixture, followed by sequential dialysis, and G100 chromatography monitored by SDS-polyacrylamide gel electrophoresis (6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Troponin binds very tightly to the thin filament, making the affinity difficult to measure directly. Therefore, the effect of the mutation on this process was determined by competition (21Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274 (; Correction (1999) J. Biol. Chem.274, 31750): 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Unlabeled control or mutant troponin was used to displace radiolabeled control troponin from the thin filament. Increasing concentrations of unlabeled troponin were added to labeled thin filaments, and displacement was measured by determining the supernatant radioactivity after thin filament sedimentation in a TLA100 ultracentrifuge at 35,000 rpm for 30 min. Data were analyzed as in Hinkle et al. (21Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274 (; Correction (1999) J. Biol. Chem.274, 31750): 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), to determine the value for KR, i.e.the ratio of the affinity of the competing troponin for the thin filament, relative to the thin filament affinity of control [3H]troponin. Conditions: 25 °C, 7 μmactin, 7 μm myosin S1, 3 μm tropomyosin, 1 μm3H-labeled troponin, 10 mmTris (pH 7.5), 300 mm KCl, 3 mmMgCl2, 0.2 mm dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.5 mm EGTA, and either 0 or 0.6 mm CaCl2. These high ionic strength conditions were used to impair troponin-tropomyosin polymerization, which otherwise interferes with binding measurements (22Hill L.E. Mehegan J.P. Butters C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: 16106-16113Abstract Full Text PDF PubMed Google Scholar). Competing unlabeled troponin was added to samples at concentrations ranging between 0 and 4 μm. Thin filaments were reconstituted by mixing F-actin (24 μm) first with cardiac tropomyosin (8 μm) and then troponin (8 μm, prepared as above from wild type troponin I and C and mutant TnT) in a solution of 250 mm KCl (used to prevent thin filament aggregation that tends to be induced by troponin), 3 mm MgCl2, 0.5 mm EGTA, 1 mm dithiothreitol, 10 mm sodium phosphate buffer (pH 7.1). Filaments were allowed to incubate at room temperature (∼25 °C) for 5–10 min before making a 20-fold dilution with additional buffer lacking KCl such that the final KCl concentration was 12.5 mm. Samples of reconstituted filaments were also treated with Ca2+ by a comparable 20-fold dilution in the same buffer lacking both KCl and EGTA but containing 0.1 mmCaCl2. Diluted filaments were then applied to carbon-coated electron microscope grids and negatively stained as described previously (23Moody C. Lehman W. Craig R. J. Muscle Res. Cell Motil. 1990; 11: 176-185Crossref PubMed Scopus (59) Google Scholar). Electron micrograph images were recorded on a Philips CM120 electron microscope at × 60,000 magnification under low dose conditions (∼12 e−/Å2). Micrographs were digitized using either Eikonix model 1412 or Imacon Flextight Precision II scanners at a pixel size corresponding to 0.7 nm in the filaments. Regions of filaments were selected and straightened as described previously (24Hodgkinson J.L. El-Mezgueldi M. Craig R. Vibert P. Marston S.B. Lehman W. J. Mol. Biol. 1997; 273: 150-159Crossref PubMed Scopus (62) Google Scholar, 25Egelman E.H. Ultramicroscopy. 1986; 19: 367-374Crossref PubMed Scopus (93) Google Scholar). Helical reconstruction was carried out by standard methods (26DeRosier D.J. Moore P.B. J. Mol. Biol. 1970; 52: 355-369Crossref PubMed Scopus (408) Google Scholar, 27Amos L.A. Klug A. J. Mol. Biol. 1975; 99: 51-73Crossref PubMed Scopus (122) Google Scholar, 28Owen C. Morgan D.G. DeRosier D.J. J. Struct. Biol. 1996; 116: 167-175Crossref PubMed Scopus (86) Google Scholar) as described previously (10Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar,29Vibert P. Craig R. Lehman W. J. Cell Biol. 1993; 123: 313-321Crossref PubMed Scopus (62) Google Scholar). While actin and tropomyosin contributions are readily delineated in reconstructions, densities due to troponin are not apparent (see Ref. 30Milligan R.A. Whittaker M. Safer D. Nature. 1990; 348: 217-221Crossref PubMed Scopus (321) Google Scholar). Resolution (31Owen C. DeRosier D. J. Cell Biol. 1993; 123: 337-344Crossref PubMed Scopus (79) Google Scholar) in all reconstructions was between 2.5 and 3.0 nm; comparison of reconstructions made from images digitized on the respective scanners showed no obvious differences at this resolution. Tropomyosin and actin densities displayed in reconstructions were significant (32Milligan R.A. Flicker P.F. J. Cell Biol. 1987; 105: 29-39Crossref PubMed Scopus (200) Google Scholar, 33Trachtenburg S. DeRosier D.J. J. Mol. Biol. 1987; 195: 571-601Google Scholar) at equal to or greater than 99.95% confidence levels. To measure myosin S1-ADP binding to control and mutant thin filaments, actin was labeled on Cys374 withN-(1-pyrenyl)iodoacetamide, which is sensitive to bound S1 (34Criddle A.H. Geeves M.A. Jeffries T. Biochem. J. 1985; 232: 343-349Crossref PubMed Scopus (179) Google Scholar). Steady state fluorescence intensity was monitored during titration of myosin S1 in 1.8-ml stirred, water-jacketed samples at 25 °C. Excitation and emission wavelengths were set at 368 and 407 nm, respectively, using an SLM 8000 spectrofluorometer. The conditions were 1 μm actin, 0.4 μm tropomyosin, 0.4 μm control or mutant troponin, 20 mmimidazole (pH 7.5), 150 mm KCl, 3 mmMgCl2, 2 mm ADP, 0.2 mg/ml bovine serum albumin, 25 units of hexokinase, 1 mm glucose, 20 μmP1,P5-di(adenosine 5′)-pentaphosphate, 0.5 mm EGTA, with or without CaCl2 added to 0.6 mm. Fluorescence data were analyzed as in Ref. 14Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, with an 80% decrease in fluorescence representing 100% saturation of actin with myosin. Data were modeled as described previously (35Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), to estimate the effect of the mutation on the equilibria between switched on and off states. In consideration of Table I, this analysis assumed that the mutation selectively alters the free energy for formation of the myosin-blocking state and the Ca2+ state of the thin filament, to degrees determined by curve-fitting of the myosin S1 binding data. All other parameters (35Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) were held constant.Table IEffect of a cardiomyopathy-inducing TnT mutation on troponin binding to thin filamentsConditionControl troponinTroponin (Δex 15,16)pCa 4, myosin S10.95 ± 0.131-aPresent study, average of four determinations using native troponin and one determination using reconstituted troponin.0.71 ± 0.051-bPresent study, average of four determinations.pCa 4, no myosin1.03 ± 0.061-cFrom Ref. 6.0.43 ± 0.021-cFrom Ref. 6.pCa > 8, no myosin1.1 ± 0.11-cFrom Ref. 6.0.22 ± 0.031-cFrom Ref. 6.The tabulated values are measurements of KR: the affinity of troponin for actin-tropomyosin thin filaments, relative to the affinity of wild type troponin labeled with [3H]iodoacetic acid on TnT Cys39. Results are based upon competitive binding experiments as shown in representative data in Fig. 1.1-a Present study, average of four determinations using native troponin and one determination using reconstituted troponin.1-b Present study, average of four determinations.1-c From Ref. 6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar. Open table in a new tab The tabulated values are measurements of KR: the affinity of troponin for actin-tropomyosin thin filaments, relative to the affinity of wild type troponin labeled with [3H]iodoacetic acid on TnT Cys39. Results are based upon competitive binding experiments as shown in representative data in Fig. 1. Ca2+ controls muscle contraction by reversibly binding to the globular domain of troponin, which includes TnC, TnI, and a portion of TnT that contains the 28 residues removed by the FHC splice site mutation (reviewed in Ref. 36Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (456) Google Scholar). The interaction of troponin's globular domain with actin and tropomyosin is Ca2+-sensitive and is believed crucial for regulation. Previously, we showed that truncation of TnT's 28 COOH-terminal residues weakens troponin binding to thin filaments (6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In the absence of Ca2+, the mutant troponin has only 22% the normal affinity for the thin filament, and in the presence of Ca2+ its affinity is 43% that of control troponin (TableI) (6Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We show here that, in contrast, the mutation has little effect on troponin binding when myosin is also bound to the thin filament (Fig. 1). In averages of multiple experiments such as that shown in Fig. 1, binding of troponin to filaments decorated with myosin subfragment 1 (S1) was only slightly diminished by the FHC TnT mutation. The affinity was almost 75% that of the labeled control troponin, considerably greater than the values obtained in the absence of myosin S1 (Table I). Thus the mutation has different effects on thin filament stability under different conditions, suggesting that it could affect the equilibrium constants among the various thin filament conformations and therefore transitions among thin filament states. Although the above measurements provide thermodynamic information on thin filament stability, they do not determine how the TnT mutation might affect filament structure. For example, the position of tropomyosin could be abnormal in the presence of the mutant TnT, especially in the absence of both Ca2+ and myosin, when troponin binding to the thin filament is particularly weak. Electron microscopy was performed to determine the structural impact of the mutant TnT on thin filaments reconstituted with otherwise normal troponin subunits and tropomyosin. Thin filaments in electron micrographs of negatively stained samples containing normal and mutant troponin (Fig.2) were well dispersed in both the presence and absence of Ca2+, so any effects were not due to possible nonspecific filament aggregation caused by the mutation. In three-dimensional reconstructions of thin filaments reconstituted using mutant TnT, the position of tropomyosin was readily identified in helical projection and cross-section (Fig.3) and in surface view (Fig.4), both in the presence and absence of Ca2+. In filaments examined in the absence of Ca2+, tropomyosin was positioned at the inner aspect of the outer domain of actin in close contact with actin subdomains 1 and 2. In contrast, in the presence of Ca2+, tropomyosin moved to the outer edge of the inner domain of actin over subdomains 3 and 4, exposing most of the actin residues believed to interact with myosin. This regulatory movement of tropomyosin was indistinguishable from that observed in our previous work with cardiac muscle thin filaments containing wild type troponin examined under Ca2+ and Ca2+-free conditions (14Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 37Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar,38Xu C. Craig R. Tobacman L.S. Horowitz R. Lehman W. Biophys. J. 1999; 77: 985-992Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Since tropomyosin was found in the normal blocking and Ca2+-induced positions in these filaments, the effects of the TnT mutation on inhibition and activation of myosin S1-thin filament MgATPase rates and on troponin-thin filament binding were not due to aberrant tropomyosin position.Figure 3Helical projections (a,b) and transverse sections (z-sections) (c, d) of maps of three-dimensional reconstructions of negatively stained F-actin tropomyosin-troponin complexes containing mutant troponin T. Helical projections were formed by projecting component densities down the long pitch actin helices (i.e. along the n = 2 helical tracks) onto a plane perpendicular to the thin filament axis; hence, the resulting projections show axially averaged positions of tropomyosin relative to actin made to appear bilaterally symmetrical about actin's central axis. In contrast, transverse sections show the position of tropomyosin at a given axial level along filaments and connectivity to specific subdomains of actin. Because adjacent actin monomers on either side of the filament axis are staggered, sectioning through the center of actin subdomains 1 and 3 of one actin monomer results in sectioning through subdomains 2 and 4 of the other.a and c, EGTA-treated filaments, actin subdomains 1–4 are labeled in c. b and d, Ca2+-treated F-actin. Note the tropomyosin density (arrows) associated with subdomains 1 and 2, i.e.on the outer domain of actin monomers of EGTA-treated filaments, and on subdomains 3 and 4, i.e. on the inner domain of in Ca2+-treated filaments. Sections shown are at the same axial position in each reconstruction, and the actin monomers in projections and sections have the same relative orientation. Reconstructions were generated by averaging a data set containing 20 EGTA-treated filaments and another having 8 Ca2+-treated filaments. The average phase residuals (Ψ ± S.D.), measuring the relative fitting between individual filaments in each set and the averaged data, were 60.1 ± 5.0° and 60.6 ± 7.2° for EGTA- and Ca2+-treated filaments, respectively. The average up-down phase residuals (ΔΨ ± S.D.), measuring relative filament polarity, were 19.5 ± 5.6° and 17.1 ± 7.0°, respectively. The densities contributing to actin and tropomyosin in the maps shown were statistically significant at confidence levels greater than 99.95%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Surface views of reconstruction of mutant thin filaments showing the positions of tropomyosin strands on actin. In EGTA (a), tropomyosin is associated with the inner edge of the outer actin domain of actin. Note the interaction of tropomyosin with actin subdomain-1 (single white arrowheads) and bridge of density over the neighboring subdomain-2, while subdomains-3 and -4 remain unobstructed (black cross). After Ca2+ treatment (b), tropomyosin is associated with the outer edge of the inner actin domain of actin. Note that tropomyosin now interacts with subdomain-3 (double white arrowheads) while bridging over subdomain-4 and that here subdomains-1 and -2 (black asterisk) are unobstructed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The above structural results leave unanswered the question of how TnT truncation alters Ca2+-sensitive regulation of cardiac contraction. To address this, the effect of the mutation on myosin S1 binding to the thin filament was examined, since Ca2+-dependent control of this process is central to how troponin and tropomyosin regulate contraction (10Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar, 36Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (456) Google Scholar,39Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1316) Google Scholar). Our results show, as shown previously, that myosin binding to control thin filaments is very cooperative in the absence of Ca2+, resulting in a sigmoidal binding curve (Fig.5, squares) (35Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 40Trybus K.M. Taylor E.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7209-7213Crossref PubMed Scopus (126) Google Scholar, 41Greene L.E. Eisenberg E. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2616-2620Crossref PubMed Scopus (217) Google Scholar). Virtually identical results were found for thin filaments containing the mutant TnT (triangles), with one important exception, namely, a much lower cooperativity in myosin bindi
Structure function
Troponin T
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Tropomyosin
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Tropomyosin
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Muscle contraction is a finely tuned mechanism involving cyclical interactions between
actin and myosin, regulated by calcium through troponin and tropomyosin and
modulated by myosin binding protein-C. Genetic mutations of the proteins involved in
such complex mechanism can thus lead to potential life threatening diseases, such as
Hypertrophic Cardiomyopathy (HCM). Although being mostly asymptomatic, HCM
affects 1 in 500 people, ultimately leading to poor prognosis and sudden death,
thought to occur through the impairment of relaxation during diastole.
In this thesis I present the experiments conducted to improve our current
understanding of the molecular mechanism behind HCM, specifically on the role of
tropomyosin and myosin binding protein-C in modulating thin filament activation and
relaxation. Using a single molecule approach, we first visualised fluorescent myosin
binding to reconstituted thin filaments and examined their dynamics in the presence
of the tropomyosin HCM causing E180G mutation, demonstrating a shift of the thin
filament activation state towards the closed state, facilitating myosin binding at low
calcium, and a reduction of the thin filament regulatory unit.
We then looked at the dynamics of very highly concentrated clusters of myosin,
showing how the sudden collapse of these active regions cannot be explained by
normal relaxation mechanisms, thus suggesting an alternative mechanistic role for
tropomyosin and how its mutations could lead to impaired relaxation in HCM.
Finally, we turned our focus on N-terminal fragments of cardiac myosin binding
protein-C (cMyBP-C) and study their role in thin filament activation, by looking at how
they affect acto-myosin interactions. We found that only the presence of the whole
cMyBP-C N-terminus was able to promote acto-myosin interactions at low Ca2+ or
repressing them at high Ca2+. Moreover, by looking at the dynamics of the fragments,
we were able to determine that cMyBP-C possesses a two steps binding mechanism to
actin, leading us to define its mechanism by which it activates the thin filament.
Tropomyosin
Meromyosin
Nebulin
Muscle relaxation
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