Structural Dynamics of Cardiac Myosin Binding Protein-C and its Myofilament Binding Partners, Detected by Site-Directed Spectroscopy

We have created a high-resolution time-resolved spectroscopy molecular biosensor for heart failure therapeutic discovery based on the structural dynamics of cardiac myosin-binding protein C (cMyBP-C). Drugs of interest mimic phosphorylation states of cMyBP-C, which influences myocardial contractility. β-adrenergic stimulation enhances contractility in myocardium, in part due to Protein Kinase A (PKA)-mediated phosphorylation of cMyBP-C. Recent structural and functional results have provided an understanding for how cMyBP-C physiologically regulates inotropy and lusiotropy in the heart by modulating actin-myosin interactions. Phosphorylation introduces a structural change within the regulatory M domain of cMyBP-C, leading to altered actin-myosin binding, and regulation of myocardial contraction. Previous molecular dynamics simulations suggested a specific rotation within the M domain upon phosphorylation that facilitates bending and exposes a putative binding site that is only accessible in the phosphorylated molecule. We hypothesized that these structural rearrangements in nanometer spatial orientation could be measured spectroscopically in mechanistic studies via introduction of site-directed probes within the M domain of cMyBP-C using time-resolved fluorescence resonance energy transfer (TR-FRET). Moreover, we hypothesized that this fluorescently-labeled recombinant muscle protein could be used as a TR-FRET biosensor for detecting potential drugs that alter the M domain structure in specific ways. These drugs will be particularly relevant to enhancement of myocardial contractility and represent novel therapeutics for heart failure and cardiomyopathy. Here, we engineered pairs of cysteine residues within the M domain for FRET labeling and tested the effects of phosphorylation to influence the tertiary structure and dynamics of cMyBP-C. We have characterized cMyBP-C’s molecular basis for fine-tuning contraction and our biosensor is well aligned for high-throughput spectroscopic screens for drugs that perturb structure specific to phosphorylation state. Compound discovery using this assay is applicable to development of novel cardiac disease therapies by enhancing contractility to alleviate dysfunction.

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Human cardiac myosin–binding protein C restricts actin structural dynamics in a cooperative and phosphorylation-sensitive manner

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.009543 ◽

2019 ◽

Vol 294 (44) ◽

pp. 16228-16240 ◽

Cited By ~ 2

Author(s):

Thomas A. Bunch ◽

Rhye-Samuel Kanassatega ◽

Victoria C. Lepak ◽

Brett A. Colson

Keyword(s):

Structural Dynamics ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Biological variation of cardiac myosin-binding protein C in healthy individuals

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2021-0306 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Bashir Alaour ◽

Torbjørn Omland ◽

Janniche Torsvik ◽

Thomas E. Kaier ◽

Marit S. Sylte ◽

...

Keyword(s):

Risk Stratification ◽

Protein C ◽

Myocardial Injury ◽

Binding Protein ◽

Biological Variation ◽

Cardiac Myosin ◽

Analytical Quality ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Abstract Objectives Cardiac myosin-binding protein C (cMyC) is a novel biomarker of myocardial injury, with a promising role in the triage and risk stratification of patients presenting with acute cardiac disease. In this study, we assess the weekly biological variation of cMyC, to examine its potential in monitoring chronic myocardial injury, and to suggest analytical quality specification for routine use of the test in clinical practice. Methods Thirty healthy volunteers were included. Non-fasting samples were obtained once a week for ten consecutive weeks. Samples were tested in duplicate on the Erenna® platform by EMD Millipore Corporation. Outlying measurements and subjects were identified and excluded systematically, and homogeneity of analytical and within-subject variances was achieved before calculating the biological variability (CVI and CVG), reference change values (RCV) and index of individuality (II). Results Mean age was 38 (range, 21–64) years, and 16 participants were women (53%). The biological variation, RCV and II with 95% confidence interval (CI) were: CVA (%) 19.5 (17.8–21.6), CVI (%) 17.8 (14.8–21.0), CVG (%) 66.9 (50.4–109.9), RCV (%) 106.7 (96.6–120.1)/−51.6 (−54.6 to −49.1) and II 0.42 (0.29–0.56). There was a trend for women to have lower CVG. The calculated RCVs were comparable between genders. Conclusions cMyC exhibits acceptable RCV and low II suggesting that it could be suitable for disease monitoring, risk stratification and prognostication if measured serially. Analytical quality specifications based on biological variation are similar to those for cardiac troponin and should be achievable at clinically relevant concentrations.

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Cardiac myosin‐binding protein C as a candidate biomarker in heart failure: rational but not revolutionary

European Journal of Heart Failure ◽

10.1002/ejhf.2126 ◽

2021 ◽

Author(s):

A. Mark Richards

Keyword(s):

Heart Failure ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Cardiac Myosin-Binding Protein C Modulates the Tuning of the Molecular Motor in the Heart

Biophysical Journal ◽

10.1529/biophysj.107.127787 ◽

2008 ◽

Vol 95 (2) ◽

pp. 720-728 ◽

Cited By ~ 21

Author(s):

Yves Lecarpentier ◽

Nicolas Vignier ◽

Patricia Oliviero ◽

Aziz Guellich ◽

Lucie Carrier ◽

...

Keyword(s):

Protein C ◽

Molecular Motor ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614020114 ◽

2017 ◽

Vol 114 (8) ◽

pp. E1355-E1364 ◽

Cited By ~ 35

Author(s):

Robert W. Kensler ◽

Roger Craig ◽

Richard L. Moss

Keyword(s):

Protein C ◽

Structural Changes ◽

Binding Protein ◽

Cardiac Contraction ◽

Cardiac Myosin ◽

Cross Bridge ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Thick Filaments ◽

Myosin Binding

Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium.

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