Crystal Structure of the C1 domain of Cardiac Myosin Binding Protein-C: Implications for Hypertrophic Cardiomyopathy

106

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C’s N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain’s extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C’s inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C’s calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C’s phosphorylation state.

Section: Resultsmentioning

confidence: 59%

Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

Previs

Mun

Michalek

et al. 2016

106

“…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: 72%

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

Rybakova

et al. 2009

143

189

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...

“…We did not find any segments with C1 bound in the side mode within those segments because all of the segments had C1 bound to F-actin in the front mode. The overall reconstruction of the C1 front class did not match the crystal structure of the C1 Ig domain (25), but subsequent sorting revealed that C1 binds to the front of the actin molecule in three structural modes (Fig. 3 A-C), which we denote here as C1 front mode 1 (C1-F1), C1 front mode 2 (C1-F2), and C1 front mode 3 (C1-F3).…”

Section: C0 Does Notmentioning

confidence: 95%

C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

Harris

Belknap

Sciver

et al. 2016

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca 2+ have been achieved. We suggest that Ca 2+ modulates the interaction of cMyBP-C with the TF in the sarcomere.