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

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0-C2) to labeled F-actin reduced rotational flexibility by 20-25°, indicated by increased final anisotropy of the TPA decay. The effects of C0-C2 on actin TPA were highly cooperative (n = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0-C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser to Asp phosphomimetic substitutions in the M-domain of C0-C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0-C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0-C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3β) site in the Pro/Ala-rich linker of C0-C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C-induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin-myosin interactions.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R00 HL122397, R01 HL141564]Open access article.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin-binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0-C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0-C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin-tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0-C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0-C2 phosphorylation and altered binding. Lastly, C0-C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery. © 2021 Bunch et al.National Institutes of HealthOpen access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating contractile dysfunction in heart failure. Mimicking the structural dynamics of phosphorylated cMyBP-C by small-molecule drug binding could lead to therapies that modulate cMyBP-C conformational states, and thereby function, to improve contractility. We have developed a human cMyBP-C biosensor capable of detecting intramolecular structural changes due to phosphorylation and mutation. Using site-directed mutagenesis and time-resolved fluorescence resonance energy transfer (TR-FRET), we substituted cysteines in cMyBP-C N-terminal domains C0 through C2 (C0-C2) for thiol-reactive fluorescent probe labeling to examine C0-C2 structure. We identified a cysteine pair that upon donor-acceptor labeling reports phosphorylation-sensitive structural changes between the C1 domain and the tri-helix bundle of the M-domain that links C1 to C2. Phosphorylation reduced FRET efficiency by ~18%, corresponding to a ~11% increase in the distance between probes and a ~30% increase in disorder between them. The magnitude and precision of phosphorylation-mediated TR-FRET changes, as quantified by the Z'-factor, demonstrate the assay's potential for structure-based high-throughput screening of compounds for cMyBP-C-targeted therapies to improve cardiac performance in heart failure. Additionally, by probing C1's spatial positioning relative to the tri-helix bundle, these findings provide new molecular insight into the structural dynamics of phosphoregulation as well as mutations in cMyBP-C. Biosensor sensitivity to disease-relevant mutations in C0-C2 was demonstrated by examination of the hypertrophic cardiomyopathy mutation R282W. The results presented here support a screening platform to identify small molecules that regulate N-terminal cMyBP-C conformational states.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]