Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

Whitten, Andrew E.; Jeffries, Cy M.; Harris, Samantha P.; Trewhella, Jill

doi:10.1073/pnas.0808903105

Cited by 114 publications

(154 citation statements)

References 41 publications

(43 reference statements)

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“…Remarkably, we discovered that all 3 truncated forms of recombinant cMyBP-C studied have dramatically different phosphorylation states even with a seemingly minor truncation compared with the full-length protein. Truncated recombinant proteins are routinely used to substitute the fulllength forms in crystal structure (46) and functional studies (47). Our study provides direct evidence of alterations in posttranslational states between truncated and full-length recombinant proteins, which can lead to variations in their structure and function.…”

mentioning

confidence: 89%

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

Rybakova

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

143

189

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Cardiac myosin binding protein C (cMyBP-C), bound to the sarcomere's myosin thick filament, plays an important role in the regulation of muscle contraction. cMyBP-C is a large multidomain protein that interacts with myosin, titin, and possibly actin. Mutations in cMyBP-C are the most common known cause of heritable hypertrophic cardiomypathies. Phosphorylation of cMyBP-C plays an essential role in the normal cardiac function. cMyBP-C (142 kDa) has 81 serine and 73 threonine residues presenting a major challenge for unequivocal identification of specific phosphorylation sites. Top-down mass spectrometry, which directly analyzes intact proteins, is a powerful technique to universally observe and quantify protein posttranslational modifications without a priori knowledge. Here, we have extended top-down electron capture dissociation mass spectrometry to comprehensively characterize mouse cMyBP-C expressed in baculovirus. We have unambiguously identified all of the phosphorylation sites in the truncated (28 -115 kDa) and full-length forms of cMyBP-C (142 kDa) and characterized the sequential phosphorylations, using a combination of top-down and middle-down (limited proteolysis) MS approach, which ensures full sequence coverage. Unit mass resolution and high mass accuracy (<5 ppm) have been achieved for a 115-kDa protein (the largest protein isotopically resolved to date). Remarkably, we discovered that truncations in recombinant proteins, even a seemingly minor one, can dramatically alter its phosphorylation state, which is significant because truncated recombinant proteins are routinely substituted for their full-length forms in crystal structure and functional studies. Our study provides direct evidence of alterations in the posttranslational state between the truncated and full-length recombinant proteins, which can lead to variations in structure and function.electron capture dissociation ͉ hypertrophic cardiomypathy C ardiac myosin binding protein C (cMyBP-C), located in the sarcomere's thick filament, plays an important role in the regulation of cardiac contraction and maintenance of myosin filament structure (1-5). Mutations in cMyBP-C are widely recognized as the most common cause of many heritable hypertrophic cardiomypathies in which the heart is hypertrophied, hypercontractile, and susceptible to electric and mechanic failure (6). cMyBP-C is a large multidomain protein including 8 IgI-like domains and 3 fibronectin (Fn) type III-like domains, numbered from the N terminus as domains C0-C10 (Fig. 1). cMyBP-C is specific to cardiac muscle owing to an additional IgI domain at the N terminus (C0), insertion of 28 residues within the IgI (C5) domain and a 9-aa-residue insert within the MyBP-C motif compared with fast/slow skeletal MyBP-C (1, 2). The C terminus of cMyBP-C binds to the thick filament backbone via interactions with myosin and titin primarily through C7-C10 (2, 7). The C1-C2 region of MyBP-C also has been shown to bind to myosin S2 segment (8) and appears to interact with actin thin filament (9).Ph...

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confidence: 89%

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

Rybakova

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

143

189

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“…The structural parameters derived from SAXS also indicated that it is possible for the N-terminal domains to reach across the interfilament space and interact with actin. Subsequent SANS 81 with contrast variation studies of the assembly formed between actin and deuterated C0C2 showed that the N-terminal domains of cMyBP-C promote filamentous actin (F-actin) assembly and decorate actin filaments in a regular fashion. The length of the average C0C2/F-actin assembly exceeded the size limit that could be determined from the SANS data (a limit determined by the minimum q-value measured) so cross-sectional structural analysis was pursued.…”

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confidence: 99%

“…81 (A) An example of the pair-distance distribution profiles of cross-section P c (r) calculated from scattering data at the solvent match points of cMyBP-C 2 H-C0C2 ($ 100% 2 H 2 O) and F-actin ($ 40% 2 H 2 O) within the macromolecular assembly in comparison with the P c (r) of undecorated F-actin. The P c (r) shows the characteristic peaks with relative intensities as expected for an extended four domain structure; the first, second, third, and forth peaks correspond to atom-pair distances within a single domain, a pair of neighboring domains, domains separated by one domain, and domains separated by two domains.…”

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confidence: 99%

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

Jeffries

Trewhella

2011

Biopolymers

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Small‐angle X‐ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small‐angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 505–516, 2011.

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“…On activation, Tn binds Ca 2+ , causing Tm to move onto actin SD3 (the "closed" position), exposing myosin binding sites on SD1 and initiating crossbridge cycling and contraction (29)(30)(31)(32)(33)(34)(35). When a model of Tm in its low Ca 2+ (blocked) state is positioned on the reconstruction of F-actin decorated with C0C3, it appears to clash with cMyBP-C's C0 and C1 domains, suggesting that cMyBP-C and Tm might compete for binding to SD1 in the relaxed thin filament (25,27,28). As a consequence, cMyBP-C might be expected to activate the thin filament by physically preventing Tm from assuming its blocked position (25,27,28).…”

mentioning

confidence: 99%

“…Neutron scattering and NMR titration analysis of F-actin decorated with the N-terminal fragment C0C2 (Fig. 1) suggested that C0C2 binds to subdomain 1 (SD1) and the DNase loop of actin (25) via key regions within C0 and C1 (10). More direct observations by negative stain electron microscopy (26) and 3D reconstruction (27) suggest that C0 and C1 bind to SD1 of actin, whereas the M domain crosses Significance Myosin-binding protein C (MyBP-C) is a component of myosin filaments, one of the two sets of contractile elements whose relative sliding is the basis of muscle contraction.…”

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confidence: 99%

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Mun

Previs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

138

207

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Significance Myosin-binding protein C (MyBP-C) is a component of myosin filaments, one of the two sets of contractile elements whose relative sliding is the basis of muscle contraction. In the heart, MyBP-C modulates contractility in response to cardiac stimulation; mutations in MyBP-C lead to cardiac disease. The mechanism by which MyBP-C modulates cardiac contraction is not understood. Using electron microscopy and a light microscopic assay for filament sliding, we demonstrate that MyBP-C binds to the other set of filaments, containing actin and the regulatory component, tropomyosin. In so doing, it displaces tropomyosin from its inhibitory position to activate actin filament interaction with myosin, promoting filament sliding. These findings provide insights into the molecular basis of heart function.

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Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

Cited by 114 publications

References 41 publications

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

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