A Gain-of-Function Mutation in the M-domain of Cardiac Myosin-binding Protein-C Increases Binding to Actin

We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electronelectron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. muscle | protein kinase A | fluorescence resonance energy transfer | DEER | molecular dynamics simulation

Section: Phosphorylation Unmasks the Charged Binding Strut Of The Tricontrasting

confidence: 54%

“…Unlike the rest of this primarily disordered domain, a segment at the motif's C-terminal end is a well-ordered triple-helix bundle ( Fig. 1B) (15)(16)(17). We hypothesize that phosphorylation-dependent structural changes in this region are critical for the function of cMyBP-C.…”

mentioning

confidence: 99%

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

Colson

Thompson

Espinoza-Fonseca

et al. 2016

“…However, despite the widespread appeal of this idea, evidence that cMyBP-C limits the mobility of myosin heads via interactions with the thick filaments is still lacking. In contrast, we and others have shown that the NTDs of the cMyBP-C (e.g., C0, PA, C1, M, and C2) can bind to the TF and potently activate actomyosin interactions (14,16,29,30). The precise mechanism(s) by which cMyBP-C affects contraction is still unresolved, in part because there is a lack of understanding of how individual domains of cMyPB-C contribute to the overall effects of this multidomain protein on muscle contraction.…”

Section: Discussionmentioning

confidence: 95%

“…Force measurements with or without C0 and C1 were performed as previously described with slight modifications (29). Treatment of all animals was in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals using protocols approved by the Institutional Animal Care and Use Committee at University of Arizona.…”

Section: Cryo-emmentioning

confidence: 99%

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

Self Cite

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.

“…Assuming that C0C1f binds to the thin filament through the same contacts as C0C2, then additional sites of actin interaction must exist beyond the first 17 amino acids of the M-domain that are present in the C0C1f fragment. For example, arginines 279/280 (further along the M-domain) have been identified as critical to actin binding (9), and when mutated to alanines, they diminish the capacity of C1C2 fragments to activate thin filaments in skinned rat trabeculae (49).…”

Section: Discussionmentioning

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

148

207

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.