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Myosin-Binding Protein-C: Bridging the Gap
Commentary
Myosin-Binding Protein-C: Bridging the Gap
Gerald Offer Muscle Contraction Group, School of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, UK
Correspondence to :[email protected] DOI of the original article: http://dx.doi.org/10.1016/j.jmb.2014.10.023 http://dx.doi.org/10.1016/j.jmb.2014.11.006 Edited by J. Sellers
Recent progress in determining the structural organisation of the accessory protein, myosin-binding protein-C (MyBP-C), in vertebrate muscle thick filaments has set the stage for a deeper understanding of how it functions. The structure and function of MyBP-C have been extensively researched [1–8], spurred on by the finding that mutations in MyBP-C are a leading cause of inherited hypertrophic cardiomyopathy [4,9–13]. MyBP-C was discovered over 40 years ago as an impurity in preparations of myosin [14]. The molecule is a flexible rod [14–17] about 40 nm long and 3–4 nm wide made up of an end-to-end array of fibronectin-like and immunoglobulin-like domains [18]. Three different isoforms encoded by different genes are present in fast skeletal, slow skeletal and cardiac muscle. At their N-terminus, the skeletal isoforms have a motif rich in proline and alanine residues followed by 10 domains labelled C1 to C10 with a flexible but compact m-domain between C1 and C2. The cardiac isoform has an additional domain C0 at the N-terminus and an insertion in the m-domain that gives it regulatory properties by enabling four serine residues to be phosphorylated in response to adrenergic stimulation [19].
Binding Partners MyBP-C binds to both myosin and titin, the two main components of the thick filament [14,20,21]. There are two distinct binding sites in the myosin tail, one halfway along the light meromyosin region [22,23] and one on the first 126 residues of the subfragment-2 region [24,25]. In titin strands, the eleven 11-domain super-repeats provide the binding sites for MyBP-C and other accessory proteins [21]. Expression of individual domains, or small groups of domains, shows that C10 binds light meromyosin strongly [26] and C8–C10 bind less strongly to titin 0022-2836/© 2014 Elsevier Ltd. All rights reserved.
[20,21]. Thus, MyBP-C is anchored on the surface of the thick filament shaft through its C-terminal region, whilst its N-terminal region can bind both subfragment-2 and the regulatory light chain of the myosin head, the two structures straddling the head–tail junction [24,27]. The binding of MyBP-C to the head– tail junction raises the possibility that this interaction might have a regulatory function by controlling the position of myosin heads relative to the thick filament shaft [24,28]. The N-terminal region of MyBP-C also binds actin [29], raising the intriguing possibility that MyBP-C could form crosslinks between thick and thin filaments. Although at one time actin was thought to bind many proteins non-specifically, three-dimensional reconstructions of F-actin decorated with the N-terminal region of MyBP-C show that the binding is stereospecific [30], although the domain that binds is still controversial [7]. Soon after its discovery, antibodies were raised to MyBP-C and used to label rigor muscle. Electron micrographs of sections of labelled psoas muscle showed that MyBP-C was located in 7 transverse stripes spaced 43 nm apart in each half of the A-band, sharing this periodicity with myosin and titin and thus located at every third level of myosin heads [31]. In cardiac muscle, MyBP-C is located on 9 stripes [32]. The stripes bearing MyBP-C coincide in position with the outermost 7–9 of 11 narrow (~ 8 nm) transverse stripes seen in sections of muscle or in negatively stained myofibrils or A-segments [33]. The 3-fold rotational symmetry of the thick filament suggests that there are three MyBP-C molecules at each axial position.
Orientation Whilst these studies revealed the axial distribution of MyBP-C molecules along the thick filament, their orientation remained unclear, giving no clue to their J. Mol. Biol. (2015 ) 427, 231–235
232
Commentary on Myosin-Binding Protein-C
Fig. 1. Stereo pairs of an averaged tomogram of a thick filament showing MyBP-C projections in purple mesh. Myosin heads are shown in blue and yellow. The location where actin filaments would be seen at a lower threshold is shown by the white straight lines. White curved lines indicate possible paths of the N-terminal arm of MyBP-C. (a) Side-on view and (b) filament tilted 20° towards viewer. This figure is reproduced from Luther et al. [39] with permission of Dr. Pradeep Luther and the National Academy of Sciences.
function. The presence of the narrow stripes suggested that a substantial part, possibly all, of the molecule was organised in transverse planes 43 nm apart. Specific proposals included the possibility that all or part of the MyBP-C wrapped circumferentially around the thick filament shaft [2,34,35], possibly extending radially outward [35]. However, because there were indications that the periodicity of MyBP-C might be very slightly greater (43.4 nm) than myosin, it was alternatively suggested that the molecule might be wholly axially oriented [36] or mostly axially oriented with just the N-terminal region pointing out radially and interacting with the actin filament through the proline/ alanine-rich motif [37]. More recently, electron micrographs of cardiac thick filaments showed at least three domains thought to be MyBP-C close to, but at a higher radius than, titin strands [38], indicating that this part of the molecule was axially oriented. Tomography of freeze-substituted resting frog sartorius muscle showed that the stripes of density seen in sections were formed by near-radial projections bridging the gap between the surface of the thick filament and neighbouring actin filaments [39] (Fig. 1). This was convincing evidence that MyBP-C could bind to actin in vivo as well as in vitro and strongly suggested a near-radial distribution of at least part of the MyBP-C molecule. The projections were curved and able to interact with either of two neighbouring actin filaments. To distinguish between the possibilities for the orientation of the MyBP-C domains, Lee et al. in the
featured article in this issue have labelled mouse cardiac myofibrils with polyclonal antibodies specific to the N-terminal region, the C-terminal region and the central region of the MyBP-C molecule. All labelled 9 transverse stripes but their exact axial position varied with the antibodies used. The stripes labelled by the antibodies to the C-terminal region were 10 nm nearer the M-line than those labelled by antibodies to the N-terminal region. This ruled out the possibility that the entire MyBP-C molecule was oriented parallel with the thick filament axis. Drawing on the tomograms of muscle sections and the electron micrographs of cardiac thick filaments, the results were explained by a model in which the N-terminal domains were arranged near-radially extending out to neighbouring thin filaments whilst at least three C-terminal domains were arranged parallel with the thick filament axis. The polarity of xthe polypeptide chains in this C-terminal region was thus defined; they run parallel, rather than anti-parallel, to the myosin tails. In the model, there was a sharp bend between the N-terminal and the C-terminal arms of the molecule. The model bears an interesting resemblance to the appearance of shadowed cardiac MyBP-C molecules [15] where the commonest appearance were V-shaped particles, the two arms having an angular separation of ~59 ± 19°, one arm with a length of ~ 24 nm being about 4 nm longer than the shorter arm. Small-angle X-ray scattering of fragments of MyBP-C fits in with the model by indicating that C0–C4 is extended and by suggesting that there might
Commentary on Myosin-Binding Protein-C
be flexibility between C5 and C6 [17]. It would be desirable to examine cardiac MyBP-C molecules by negative staining to test whether there is indeed a correlation with the model and whether phosphorylation changed their shape. One concern with the hypothesis that the narrowness and regularity of the MyBP-C stripes requires the N-terminal arm of MyBP-C to be stabilised by their binding to actin is that A-segments (isolated A-bands) show well-defined transverse stripes despite the absence of thin filaments [33]. This raises the possibility that some other structure helps hold the N-terminal arm of MyBP-C with a narrow axial spread. In solution, both fast skeletal MyBP-C and cardiac MyBP-C dimerise [14,17]. It is conceivable that the N-terminal arm of a MyBP-C molecule not only binds actin but also can interact with another MyBP-C molecule from a neighbouring thick filament. However, current evidence suggests that dimerisation occurs through interactions between C-terminal regions [17].
Function There has recently been much progress in understanding the function of MyBP-C. Knockout mice lacking cardiac MyBP-C retain sarcomere organisation [3,12,32] but show significant cardiac hypertrophy including heart enlargement, disorganisation of myocytes and fibrosis, as well as impaired diastolic and systolic function and a slightly reduced sensitivity to Ca 2 + . Whilst isometric tension was not significantly altered, the shortening velocity and power output were increased at lower loads. Hence, MyBP-C appears essential for normal cardiac functioning. In cardiac thick filaments from knockout mice, the interaction between some of the myosin heads was disrupted [32,38] and the intensity ratio of the X-ray equatorial reflections suggested that the myosin heads lay further out from the thick filament backbone [40]. Thus, one function of MyBP-C might be to interact with myosin heads near the head–tail junction and tether them closer to the backbone. The first indication that MyBP-C had a regulatory function in the heart was the demonstration that phosphorylation of MyBP-C increases tension in response to adrenergic stimulation [41]. A key aim has been to establish how the interaction of MyBP-C with actin or subfragment-2 is affected by phosphorylation and how this alters cross-bridge behaviour. Electron micrographs of thin filaments decorated with N-terminal fragments of MyBP-C show that they bind to a site on actin where tropomyosin strands would bind in their OFF position blocking the binding of myosin heads [42]. At low Ca 2+ concentrations, this causes the tropomyosin strands to move to the ON position thereby activating the thin filament. This
233 sensitisation of the thin filament to Ca 2+ was also demonstrated in motility studies. At low Ca 2+ concentrations, native thin filaments do not slide over a bed of myosin molecules. However, addition of N-terminal fragments of MyBP-C causes the filaments to slide, albeit slowly [42]. Conversely, at high Ca 2+ concentrations, the addition of MyBP-C fragments decreases the velocity of sliding probably due to the interaction of MyBP-C with actin producing drag force. The function of this drag force is unclear. It may act to damp down sudden, very rapid sliding, but this would presumably lower efficiency. Enzymological studies [43] complement these structural and motility studies. N-terminal fragments of MyBP-C inhibit the activation by F-actin of myosin subfragment-1 ATPase. However, when F-actin is replaced by cardiac thin filaments, there is a biphasic response; low ratios of MyBP-C activate the subfragment-1 ATPase particularly at low Ca 2 + concentrations, whilst higher ratios inhibit. Low ratios of MyBP-C are thought to activate by binding to actin and displacing the tropomyosin strands, whilst high ratios inhibit by competing with heads for actin sites. The relatively low ratio of MyBP-C to actin in muscle would be expected to cause the activating effects of MyBP-C to dominate. Further evidence that MyBP-C can exert a drag force has been obtained by measuring the velocity of sliding of short actin filaments over native cardiac thick filaments [44]. Typically, the sliding velocity was initially fast but then abruptly slowed by 45%; the distance travelled in the slower phase was similar to the length of the C-zone, the region bearing MyBP-C. The interpretation is that, in the initial fast phase, the actin filaments are moving along one end of the thick filament but braking occurs as they begin to enter the C-zone. Importantly, reduction of the phosphorylation levels in the m-domain of MyBP-C resulted in increased braking in the slow phase. This suggested that phosphorylation of MyBP-C controls contractility at least partly by reducing drag and hence increasing the velocity of contraction and power generation. All the above-mentioned results indicate that the binding of the N-terminal region of MyBP-C to actin is physiologically relevant and phosphorylation sensitive. There is also good evidence from solution studies that MyBP-C binds to subfragment-2 in solution and that the binding is abolished by phosphorylation [4,25]. Removal of this tether was speculated to allow myosin heads to adopt a higher radius [28,40] more favourable for binding actin. Additional evidence that MyBP-C can bind to subfragment-2 in muscle is that N-terminal fragments of MyBP-C bind to the A-band rather than to the I-band [25]. However, no high-resolution images showing MyBP-C binding to subfragment-2 in muscle are available. Current speculation is that the N-terminal region of MyBP-C switches between actin and subfragment-2 dependent on its phosphorylation state and whether or not it
234 is overlapped by actin [7]. A related question is what structural change occurs when resting muscle is activated. At an early stage of activation, the intensity of the X-ray meridional M1 reflection declines, indicating that the axial distribution of MyBP-C broadens [45]. A clue to the cause is that the M1 intensity in resting frog muscle decreases over a narrow range of sarcomere lengths from 2.6 μm (where the C-zone is totally overlapped by actin) to 3.0 μm (where none is overlapped). This suggests that the N-terminal arm of MyBP-C has a narrow axial breadth when it binds to actin, but when it is not bound to actin, it becomes axially broadened because either it is flexible or it binds to subfragment-2. The reduction in M1 intensity on activation might arise because, whilst remaining attached to actin, it tilts as the filaments slide slightly. Clearly, many intriguing puzzles remain, but the new information on how the MyBP-C molecule is arranged in the thick filament will speed the quest of understanding more fully how it functions.
References [1] Bennett PM, Fürst DO, Gautel M. The C-protein (myosinbinding protein C) family: regulators of contraction and sarcomere formation? Rev Physiol Biochem Pharmacol 1999;138:203–34. [2] Winegrad S. Cardiac myosin-binding protein C. Circ Res 1999;84:1117–26. [3] Korte FS, McDonald KS, Harris SP, Moss RL. Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C. Circ Res 2003;93:752–8. [4] Flashman E, Redwood C, Moolman-Smook J, Watkins H. Cardiac myosin-binding protein C: its role in physiology and disease. Circ Res 2004;94:1279–89. [5] Ackermann MA, Kontrogianni-Konstantopoulos A. Myosin binding protein-C: a regulator of actomyosin interaction in striated muscle. J Biomed Biotechnol 2011;2011. http://dx. doi.org/10.1155/2011/636403. [6] Sadayapann S, de Tombe PP. Cardiac myosin binding protein C: redefining its structure and function. Biophys Rev 2012;4:93–106. [7] Pfuhl M, Gautel M. Structure, interactions and function of the N-terminus of cardiac myosin-binding protein C (MyBP-C): who does what, with what, and to whom? J Muscle Res Cell Motil 2012;33:83–94. [8] van Dijk SJ, Bezold KL, Harris SP. Earning stripes: myosin binding protein-C interactions with actin. Pflugers Arch 2014; 466:445–50. [9] Watkins H, Conner D, Thierfelder L, Jarcho JA, MacRae C, McKenna WJ, et al. Mutations in the cardiac myosinbinding protein–C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet 1995; 11:434–7. [10] Bonne G, Carrier L, Bercovici J, Cruaud C, Richard P, Hainque B, et al. Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat Genet 1995;11:438–40.
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[11] Seidman JG, Seidman C. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell 2001;104:557–67. [12] Harris SP, Bartley CR, Hacker TA, McDonald KS, Douglas PS, Greaser ML, et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ Res 2002;90:594–601. [13] Harris SP, Lyons RG, Bezold KL. In the thick of it: HCMcausing mutations in myosin binding proteins of the thick filament. Circ Res 2011;108:751–64. [14] Offer G, Moos C, Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extraction, purification and characterization. J Mol Biol 1973;74:653–76. [15] Hartzell HC, Sale WS. Structure of C protein purified from cardiac muscle. J Cell Biol 1985;100:208–15. [16] Swan RC, Fischman DA. Electron microscopy of C-protein molecules from chicken skeletal muscle. J Muscle Res Cell Motil 1986;7:160–6. [17] Jeffries CM, Yanling L, Hynson RMG, Taylor JE, Ballesteros M, Kwan AH, et al. Human cardiac myosin-binding protein C: structural flexibility within an extended modular architecture. J Mol Biol 2011;414:735–48. [18] Einheber S, Fischman DA. Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily. Proc Natl Acad Sci USA 1990;87:2157–61. [19] Gautel M, Zuffardi O, Freiburg A, Labeit S. Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? EMBO J 1995;14:1952–60. [20] Furst DO, Vinkemeier U, Weber K. Mammalian skeletal muscle C-protein: purification form bovine muscle, binding to titin and the characterization of a full-length human cDNA. J Cell Sci 1992;102:769–78. [21] Freiburg A, Gautel M. A molecular map of the interactions between titin and myosin-binding protein C. Implications for sarcomeric assembly in familial hypertrophic cardiomyopathy. Eur J Biochem 1996;235:317–23. [22] Moos C, Offer G, Starr R, Bennett P. Interaction of C-protein with myosin, myosin rod and light meromyosin. J Mol Biol 1975;97:1–9. [23] Flashman E, Watkins H, Redwood C. Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod. Biochem J 2007;401:97–102. [24] Starr R, Offer G. The interaction of C-protein with heavy meromyosin and subfragment-2. Biochem J 1978;171: 813–6. [25] Gruen M, Gautel M. Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C. J Mol Biol 1999;286:933–49. [26] Okagaki T, Weber FE, Fischman DA, Vaughan KT, Mikawa T, Reinach FC. The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif. J Cell Biol 1993;123:619–26. [27] Ratti J, Rostkova E, Gautel M, Pfuhl M. Structure and interactions of myosin-binding protein C domain C0: cardiacspecific regulation of myosin at its neck? J Biol Chem 2011; 286:12650–8. [28] Weisberg A, Winegrad S. Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle. Proc Natl Acad Sci USA 1996;93:8999–9003. [29] Moos C, Mason CM, Besterman JM, Feng IN, Dubin JH. The binding of skeletal muscle C-protein to F-actin, and its
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[30]
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relation to the interaction of actin with myosin subfragment-1. J Mol Biol 1978;124:571–86. Mun JY, Gulick J, Robbins J, Woodhead J, Lehman W, Craig R. Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C. J Mol Biol 2011;410:214–25. Craig R, Offer G. The location of C-protein in rabbit skeletal muscle. Proc R Soc Lond B Biol Sci 1976;192:451–61. Luther PK, Bennett PM, Knupp C, Craig R, Padrón R, Harris SP, et al. Understanding the organisation and role of myosinbinding protein C in normal striated muscle by comparison with MyBP-C knockout cardiac muscle. J Mol Biol 2008;384:60–72. Craig R. Structure of A-segments from frog and rabbit skeletal muscle. J Mol Biol 1977;109:69–81. Offer G. C-protein and the periodicity in the thick filaments of vertebrate skeletal muscle. Cold Spring Harbor Symp Quant Biol 1973;37:87–93. Flashman E, Korkie L, Watkins H, Redwood C, MoolmanSmook JC. Support for a trimeric collar of myosin-binding protein C in cardiac and fast skeletal muscle, but not in slow skeletal muscle. FEBS Lett 2008;582:434–8. Squire JM, Harford JJ, Edman AC, Sjostrom M. Fine structure of the A band in cryo-sections. III. Crossbridge distribution and the axial structure of the human C-zone. J Mol Biol 1982;155:467–94. Squire JM, Luther PK, Knupp C. Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain. J Mol Biol 2003;331:713–24. Zoghbi ME, Woodhead JL, Moss RL, Craig R. Threedimensional structure of vertebrate cardiac muscle myosin filaments. Proc Natl Acad Sci USA 2008;105:2386–90.
235 [39] Luther PK, Winkler H, Taylor K, Zoghbi MA, Craig R, Padròn R, et al. Direct visualisation of myosin-binding protein C bridging myosin and actin filament in intact muscle. Proc Natl Acad Sci USA 2011;108:11423–8. [40] Colson BA, Bekyarova T, Fitzsimons DP, Irving TC, Moss RL. Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C. J Mol Biol 2007;367:36–41. [41] Jeacocke S, England P. Phosphorylation of a myofibrillar protein of Mr 150000 in perfused rat heart, and the tentative identification of this as C-protein. FEBS Lett 1980;122:129–32. [42] Mun JY, Previs MJ, Yu HY, Gulick J, Tobacman LS, Beck Previs S, et al. Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Proc Natl Acad Sci USA 2014;111:2170–5. [43] Belknap B, Harris SP, White HD. Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal fragments of cardiac myosin binding protein-C. Biochemistry 2014;53: 6717–24. [44] Previs MJ, Previs B, Gulick J, Robbins J, Warshaw DM. Molecular mechanics of cardiac myosin-binding protein C in native thick filaments. Science 2012;337:1215–8. [45] Reconditi M, Brunello E, Fusi L, Linari M, Martinez MF, Lombardi V, et al. Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog. J Physiol 2014;592:1119–37.
Myosin-Binding Protein-C: Bridging the Gap
Gerald Offer Muscle Contraction Group, School of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, UK
Correspondence to :[email protected] DOI of the original article: http://dx.doi.org/10.1016/j.jmb.2014.10.023 http://dx.doi.org/10.1016/j.jmb.2014.11.006 Edited by J. Sellers
Recent progress in determining the structural organisation of the accessory protein, myosin-binding protein-C (MyBP-C), in vertebrate muscle thick filaments has set the stage for a deeper understanding of how it functions. The structure and function of MyBP-C have been extensively researched [1–8], spurred on by the finding that mutations in MyBP-C are a leading cause of inherited hypertrophic cardiomyopathy [4,9–13]. MyBP-C was discovered over 40 years ago as an impurity in preparations of myosin [14]. The molecule is a flexible rod [14–17] about 40 nm long and 3–4 nm wide made up of an end-to-end array of fibronectin-like and immunoglobulin-like domains [18]. Three different isoforms encoded by different genes are present in fast skeletal, slow skeletal and cardiac muscle. At their N-terminus, the skeletal isoforms have a motif rich in proline and alanine residues followed by 10 domains labelled C1 to C10 with a flexible but compact m-domain between C1 and C2. The cardiac isoform has an additional domain C0 at the N-terminus and an insertion in the m-domain that gives it regulatory properties by enabling four serine residues to be phosphorylated in response to adrenergic stimulation [19].
Binding Partners MyBP-C binds to both myosin and titin, the two main components of the thick filament [14,20,21]. There are two distinct binding sites in the myosin tail, one halfway along the light meromyosin region [22,23] and one on the first 126 residues of the subfragment-2 region [24,25]. In titin strands, the eleven 11-domain super-repeats provide the binding sites for MyBP-C and other accessory proteins [21]. Expression of individual domains, or small groups of domains, shows that C10 binds light meromyosin strongly [26] and C8–C10 bind less strongly to titin 0022-2836/© 2014 Elsevier Ltd. All rights reserved.
[20,21]. Thus, MyBP-C is anchored on the surface of the thick filament shaft through its C-terminal region, whilst its N-terminal region can bind both subfragment-2 and the regulatory light chain of the myosin head, the two structures straddling the head–tail junction [24,27]. The binding of MyBP-C to the head– tail junction raises the possibility that this interaction might have a regulatory function by controlling the position of myosin heads relative to the thick filament shaft [24,28]. The N-terminal region of MyBP-C also binds actin [29], raising the intriguing possibility that MyBP-C could form crosslinks between thick and thin filaments. Although at one time actin was thought to bind many proteins non-specifically, three-dimensional reconstructions of F-actin decorated with the N-terminal region of MyBP-C show that the binding is stereospecific [30], although the domain that binds is still controversial [7]. Soon after its discovery, antibodies were raised to MyBP-C and used to label rigor muscle. Electron micrographs of sections of labelled psoas muscle showed that MyBP-C was located in 7 transverse stripes spaced 43 nm apart in each half of the A-band, sharing this periodicity with myosin and titin and thus located at every third level of myosin heads [31]. In cardiac muscle, MyBP-C is located on 9 stripes [32]. The stripes bearing MyBP-C coincide in position with the outermost 7–9 of 11 narrow (~ 8 nm) transverse stripes seen in sections of muscle or in negatively stained myofibrils or A-segments [33]. The 3-fold rotational symmetry of the thick filament suggests that there are three MyBP-C molecules at each axial position.
Orientation Whilst these studies revealed the axial distribution of MyBP-C molecules along the thick filament, their orientation remained unclear, giving no clue to their J. Mol. Biol. (2015 ) 427, 231–235
232
Commentary on Myosin-Binding Protein-C
Fig. 1. Stereo pairs of an averaged tomogram of a thick filament showing MyBP-C projections in purple mesh. Myosin heads are shown in blue and yellow. The location where actin filaments would be seen at a lower threshold is shown by the white straight lines. White curved lines indicate possible paths of the N-terminal arm of MyBP-C. (a) Side-on view and (b) filament tilted 20° towards viewer. This figure is reproduced from Luther et al. [39] with permission of Dr. Pradeep Luther and the National Academy of Sciences.
function. The presence of the narrow stripes suggested that a substantial part, possibly all, of the molecule was organised in transverse planes 43 nm apart. Specific proposals included the possibility that all or part of the MyBP-C wrapped circumferentially around the thick filament shaft [2,34,35], possibly extending radially outward [35]. However, because there were indications that the periodicity of MyBP-C might be very slightly greater (43.4 nm) than myosin, it was alternatively suggested that the molecule might be wholly axially oriented [36] or mostly axially oriented with just the N-terminal region pointing out radially and interacting with the actin filament through the proline/ alanine-rich motif [37]. More recently, electron micrographs of cardiac thick filaments showed at least three domains thought to be MyBP-C close to, but at a higher radius than, titin strands [38], indicating that this part of the molecule was axially oriented. Tomography of freeze-substituted resting frog sartorius muscle showed that the stripes of density seen in sections were formed by near-radial projections bridging the gap between the surface of the thick filament and neighbouring actin filaments [39] (Fig. 1). This was convincing evidence that MyBP-C could bind to actin in vivo as well as in vitro and strongly suggested a near-radial distribution of at least part of the MyBP-C molecule. The projections were curved and able to interact with either of two neighbouring actin filaments. To distinguish between the possibilities for the orientation of the MyBP-C domains, Lee et al. in the
featured article in this issue have labelled mouse cardiac myofibrils with polyclonal antibodies specific to the N-terminal region, the C-terminal region and the central region of the MyBP-C molecule. All labelled 9 transverse stripes but their exact axial position varied with the antibodies used. The stripes labelled by the antibodies to the C-terminal region were 10 nm nearer the M-line than those labelled by antibodies to the N-terminal region. This ruled out the possibility that the entire MyBP-C molecule was oriented parallel with the thick filament axis. Drawing on the tomograms of muscle sections and the electron micrographs of cardiac thick filaments, the results were explained by a model in which the N-terminal domains were arranged near-radially extending out to neighbouring thin filaments whilst at least three C-terminal domains were arranged parallel with the thick filament axis. The polarity of xthe polypeptide chains in this C-terminal region was thus defined; they run parallel, rather than anti-parallel, to the myosin tails. In the model, there was a sharp bend between the N-terminal and the C-terminal arms of the molecule. The model bears an interesting resemblance to the appearance of shadowed cardiac MyBP-C molecules [15] where the commonest appearance were V-shaped particles, the two arms having an angular separation of ~59 ± 19°, one arm with a length of ~ 24 nm being about 4 nm longer than the shorter arm. Small-angle X-ray scattering of fragments of MyBP-C fits in with the model by indicating that C0–C4 is extended and by suggesting that there might
Commentary on Myosin-Binding Protein-C
be flexibility between C5 and C6 [17]. It would be desirable to examine cardiac MyBP-C molecules by negative staining to test whether there is indeed a correlation with the model and whether phosphorylation changed their shape. One concern with the hypothesis that the narrowness and regularity of the MyBP-C stripes requires the N-terminal arm of MyBP-C to be stabilised by their binding to actin is that A-segments (isolated A-bands) show well-defined transverse stripes despite the absence of thin filaments [33]. This raises the possibility that some other structure helps hold the N-terminal arm of MyBP-C with a narrow axial spread. In solution, both fast skeletal MyBP-C and cardiac MyBP-C dimerise [14,17]. It is conceivable that the N-terminal arm of a MyBP-C molecule not only binds actin but also can interact with another MyBP-C molecule from a neighbouring thick filament. However, current evidence suggests that dimerisation occurs through interactions between C-terminal regions [17].
Function There has recently been much progress in understanding the function of MyBP-C. Knockout mice lacking cardiac MyBP-C retain sarcomere organisation [3,12,32] but show significant cardiac hypertrophy including heart enlargement, disorganisation of myocytes and fibrosis, as well as impaired diastolic and systolic function and a slightly reduced sensitivity to Ca 2 + . Whilst isometric tension was not significantly altered, the shortening velocity and power output were increased at lower loads. Hence, MyBP-C appears essential for normal cardiac functioning. In cardiac thick filaments from knockout mice, the interaction between some of the myosin heads was disrupted [32,38] and the intensity ratio of the X-ray equatorial reflections suggested that the myosin heads lay further out from the thick filament backbone [40]. Thus, one function of MyBP-C might be to interact with myosin heads near the head–tail junction and tether them closer to the backbone. The first indication that MyBP-C had a regulatory function in the heart was the demonstration that phosphorylation of MyBP-C increases tension in response to adrenergic stimulation [41]. A key aim has been to establish how the interaction of MyBP-C with actin or subfragment-2 is affected by phosphorylation and how this alters cross-bridge behaviour. Electron micrographs of thin filaments decorated with N-terminal fragments of MyBP-C show that they bind to a site on actin where tropomyosin strands would bind in their OFF position blocking the binding of myosin heads [42]. At low Ca 2+ concentrations, this causes the tropomyosin strands to move to the ON position thereby activating the thin filament. This
233 sensitisation of the thin filament to Ca 2+ was also demonstrated in motility studies. At low Ca 2+ concentrations, native thin filaments do not slide over a bed of myosin molecules. However, addition of N-terminal fragments of MyBP-C causes the filaments to slide, albeit slowly [42]. Conversely, at high Ca 2+ concentrations, the addition of MyBP-C fragments decreases the velocity of sliding probably due to the interaction of MyBP-C with actin producing drag force. The function of this drag force is unclear. It may act to damp down sudden, very rapid sliding, but this would presumably lower efficiency. Enzymological studies [43] complement these structural and motility studies. N-terminal fragments of MyBP-C inhibit the activation by F-actin of myosin subfragment-1 ATPase. However, when F-actin is replaced by cardiac thin filaments, there is a biphasic response; low ratios of MyBP-C activate the subfragment-1 ATPase particularly at low Ca 2 + concentrations, whilst higher ratios inhibit. Low ratios of MyBP-C are thought to activate by binding to actin and displacing the tropomyosin strands, whilst high ratios inhibit by competing with heads for actin sites. The relatively low ratio of MyBP-C to actin in muscle would be expected to cause the activating effects of MyBP-C to dominate. Further evidence that MyBP-C can exert a drag force has been obtained by measuring the velocity of sliding of short actin filaments over native cardiac thick filaments [44]. Typically, the sliding velocity was initially fast but then abruptly slowed by 45%; the distance travelled in the slower phase was similar to the length of the C-zone, the region bearing MyBP-C. The interpretation is that, in the initial fast phase, the actin filaments are moving along one end of the thick filament but braking occurs as they begin to enter the C-zone. Importantly, reduction of the phosphorylation levels in the m-domain of MyBP-C resulted in increased braking in the slow phase. This suggested that phosphorylation of MyBP-C controls contractility at least partly by reducing drag and hence increasing the velocity of contraction and power generation. All the above-mentioned results indicate that the binding of the N-terminal region of MyBP-C to actin is physiologically relevant and phosphorylation sensitive. There is also good evidence from solution studies that MyBP-C binds to subfragment-2 in solution and that the binding is abolished by phosphorylation [4,25]. Removal of this tether was speculated to allow myosin heads to adopt a higher radius [28,40] more favourable for binding actin. Additional evidence that MyBP-C can bind to subfragment-2 in muscle is that N-terminal fragments of MyBP-C bind to the A-band rather than to the I-band [25]. However, no high-resolution images showing MyBP-C binding to subfragment-2 in muscle are available. Current speculation is that the N-terminal region of MyBP-C switches between actin and subfragment-2 dependent on its phosphorylation state and whether or not it
234 is overlapped by actin [7]. A related question is what structural change occurs when resting muscle is activated. At an early stage of activation, the intensity of the X-ray meridional M1 reflection declines, indicating that the axial distribution of MyBP-C broadens [45]. A clue to the cause is that the M1 intensity in resting frog muscle decreases over a narrow range of sarcomere lengths from 2.6 μm (where the C-zone is totally overlapped by actin) to 3.0 μm (where none is overlapped). This suggests that the N-terminal arm of MyBP-C has a narrow axial breadth when it binds to actin, but when it is not bound to actin, it becomes axially broadened because either it is flexible or it binds to subfragment-2. The reduction in M1 intensity on activation might arise because, whilst remaining attached to actin, it tilts as the filaments slide slightly. Clearly, many intriguing puzzles remain, but the new information on how the MyBP-C molecule is arranged in the thick filament will speed the quest of understanding more fully how it functions.
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