BACKGROUND:Lmods (leiomodins) are critical for the assembly and maintenance of thin filaments in striated muscles by allowing thin filament elongation at the pointed ends. Lmod2's elongation function has been linked to both actin-binding sites (ABSs) 2 and 3, while the existence and function of an N-terminal ABS1 has been debated. METHODS:To elucidate the little-known role of Lmod2's ABS1, we created a mutant (F64D/L69D/W72D/W73D: Lmod2-quadruple mutant) predicted to decrease the binding of ABS1 to actin. We analyzed the effect of the mutations using several in vitro, cellular, and in vivo assays. RESULTS:By disrupting the interaction of Lmod2 ABS1 with actin in isolated cardiomyocytes and in mice, we engineered a super Lmod2 that results in remarkably longer thin filaments. Structural analysis determined that ABS1 of Lmod2 binds to actin through a disordered region and an amphipathic α-helix. Analysis of the mutated ABS1 revealed that the helix is destroyed, and binding to actin is maintained only in the N-terminal disordered region of Lmod2 ABS1. CONCLUSIONS:These discoveries support a model of controlled thin filament pointed end elongation by Lmod2 and provide the first direct evidence of, as well as the structural and functional mechanistic basis for, Lmod2's physiological leaky cap activity.

Mutations in cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Although most cMyBP-C mutations produce truncated proteins and cause HCM via haploinsufficiency, the mechanisms by which missense mutations result in disease remain poorly understood. Here, we have evaluated three mutations in immunoglobulin-like domains C1 (P161S, Y237S) and C2 (P371R), predicted to be pathogenic for HCM, assessing their effects on cMyBP-C actin-binding function, protein thermal stability, and residue mobility. Using a fluorescence lifetime-based actin-binding assay, we found that N-terminal mutants P161S, Y237S, and P371R enhanced C0-C2 interactions with actin in both unphosphorylated and phosphorylated states, suggesting that the mutations strengthen actin binding and make the binding resistant to phosphorylation-mediated regulation. Differential scanning calorimetry revealed that mutants exhibit destabilized thermal melting profiles with reduced unfolding temperature, energy, and cooperativity. Molecular dynamics simulations indicated that these mutations induce allosteric effects, increasing fluctuations of unstructured loops in C1 or C2 that contain key actin-binding residues. These alterations in protein stability and residue mobility may promote domains to visit binding-competent conformations more frequently, reduce the energetic cost of complex formation, and/or expose actin-interacting interfaces, thereby enhancing C0-C2 binding and contributing to HCM pathogenesis.

N-terminal cardiac myosin-binding protein C (cMyBP-C) domains (C0-C2) bind to thick (myosin) and thin (actin) filaments to coordinate contraction and relaxation of the heart. These interactions are regulated by phosphorylation of the M-domain situated between domains C1 and C2. In cardiomyopathies and heart failure, phosphorylation of cMyBP-C is significantly altered. We aimed to investigate how cMyBP-C interacts with myosin and actin. We developed complementary, high-throughput, C0-C2 FRET-based binding assays for myosin and actin to characterize the effects due to 5 HCM-linked variants or functional mutations in unphosphorylated and phosphorylated C0-C2. The assays indicated that phosphorylation decreases binding to both myosin and actin, whereas the HCM mutations in M-domain generally increase binding. The effects of mutations were greatest in phosphorylated C0-C2, and some mutations had a larger effect on actin than myosin binding. Phosphorylation also altered the spatial relationship of the probes on C0-C2 and actin. The magnitude of these structural changes was dependent on C0-C2 probe location (C0, C1, or M-domain). We conclude that binding can differ between myosin and actin due to phosphorylation or mutations. Additionally, these variables can change the mode of binding, affecting which of the interactions in cMyBP-C N-terminal domains with myosin or actin take place. The opposite effects of phosphorylation and M-domain mutations is consistent with the idea that cMyBP-C phosphorylation is critical for normal cardiac function. The precision of these assays is indicative of their usefulness in high-throughput screening of drug libraries for targeting cMyBP-C as therapy.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of smallmolecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Cardiac muscle contraction is regulated by Ca2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z′ factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.

Cardiac myosin binding protein-C (MyBP-C) accounts for about half of hypertrophic cardiomyopathy (HCM) mutations, and in the events where another sarcomeric protein is mutated, MyBP-C phosphorylation is frequently altered. MyBP-C is thought to regulate normal cardiac contractility through its interactions with actin and myosin, and this regulation is dependent on phosphorylation. Efforts in elucidating the details of MyBP-C interactions with myosin and actin have been limited due to low-throughput and labor-intensive assays.

Cardiac muscle contraction is initiated by Ca2+-induced structural changes of the thin filaments to permit cross-bridge formation. Ca2+ binding to cardiac troponin C (TnC) on actin permits myosin binding and ATP binding to myosin allows for cross-bridge cycling during contraction. Site-directed spectroscopic probes attached to TnC at position T53C have been useful to determine structure-function relationships and Ca2+ binding kinetics in thin filaments and myofibrils by monitoring fluorescence intensity. This approach would also be useful to screen for drugs that modulate TnC structure in myofibrils to elicit therapeutic functional changes for treating contractile dysfunction in cardiac disease. However, fluorescence intensity measurements in myofibrils exchanged with TnC probes are inherently prone to signal imprecision due to variable pipetting, nonhomogeneous troponin labeling/exchange, and variable sizes of myofibril preparations. In contrast, time-resolved fluorescence (TR-F, i.e., fluorescence lifetime) is largely insensitive to changes in intensity and exhibits ∼20-fold improvement in assay precision. Therefore, we have developed a lifetime-based assay using fluorescently-labeled TnC exchanged into porcine cardiac myofibrils to determine the sensitivity and precision of detecting structural changes in TnC due to physiological and therapeutic effectors of thick and thin filament function. First, we confirmed that lifetime was indeed superior to intensity measurements. We then determined the effects of Ca2+ to bind TnC and activate the thin filament and ATP to bind myosin and activate the thick filament in mock high-throughput screens. These screening assays were determined to be of excellent quality as indicated by the Z′ factor. Finally, we evaluated effects of myosin binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. We conclude that TnC lifetime-based probes allow for precise high-throughput evaluation of thick and thin filament effectors in functioning myofibrils.

Myosin binding protein-C (cMyBP-C) is a sarcomeric protein responsible for normal contraction and relaxation of the heart. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to resolve the interactions of cardiac myosin and F-actin with cMyBP-C, focusing on the N-terminal region. The results imply roles of these bound protein complexes in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure and hypertrophic cardiomyopathy (HCM). N-terminal cMyBP-C domains C0 through C2 (C0-C2) contain binding regions for interactions with both thick (myosin) and thin (actin) filaments. Phosphorylation by protein kinase A (PKA) in the cMyBP-C motif (M-domain) regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C and either myosin or actin. We engineered intermolecular pairs of labeling sites between donor-labeled myosin regulatory light chain (V105C) or F-actin (C374) and cMyBP-C (S85C in C0, C249 in C1, or P330C in M-domain) to detect interactions. Phosphorylation reduced the interaction of cMyBP-C to both myosin and actin. Further insight was gained from evaluating cMyBP-C HCM mutations T59A, R282W, E334K, and L349R, which revealed increases in myosin-FRET, increases or decreases in actin-FRET, and perturbations of phosphorylation effects. These findings elucidate binding of cMyBP-C to myosin or actin under physiological and pathological conditions, providing new molecular insight into the modulatory role of these protein-protein interactions in cardiac muscle contractility. Further, these findings suggest that the TR-FRET assays are suitable for rapid and accurate determination of quantitative binding for screening physiological conditions and compounds that affect cMyBP-C interactions with myosin or F-actin for therapeutic discovery. Significance Statement Hypertrophic cardiomyopathy (HCM) is a heritable heart disease involving mutations in genes encoding cardiac muscle proteins. Investigating the underlying molecular mechanisms of HCM mutations provides critical insight into the clinical outcomes and can translate into life-saving therapies. A leading cause of inherited HCM are mutations found in cardiac myosin binding protein-C (cMyBP-C), which binds to both myosin and actin to finely-tune contractility. Efforts in elucidating the details of cMyBP-C interactions with myosin and actin have been limited due to standard techniques that are low-throughput and labor-intensive. We have developed a set of Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays that report the phosphorylation-sensitive binding of N-terminal cMyBP-C to myosin or actin in a high-throughput plate reader format. We detect altered binding due to phosphorylation and unique changes in HCM mutant cMyBP-C binding to myosin versus actin. Our results are informative for developing precision medicine screening assays and new therapies for HCM.

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.

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.

Cardiac Myosin Binding Protein-C (cMyBP-C) is an important regulator of myocardial contraction and cross-bridge disposition; however, its contributions to thick and thin filament structural dynamics is unknown. Therefore, we performed time-resolved fluorescence (TR-F) experiments to quantify lifetime changes of environmentally-sensitive probes introduced in myofibrils isolated from ventricles of wild-type (WT) and cMyBP-C null (cMyBP-C-/-) mice. Mutant ventricular regulatory light chain (RLC V105C) labeled with CPM or cardiac troponin C (cTnC T53C) labeled with IANBD were exchanged in skinned myofibrils to replace the endogenous proteins. ATPase measurements demonstrated that the exchanges did not affect contractile function. TR-F measurements revealed differences in lifetimes of both RLC and cTnC between WT and cMyBP-C-/- myofibrils. PKA-mediated phosphorylation of myofibrils also affected lifetimes with greater effects observed in WT as compared to cMyBP-C-/-. For the RLC probe, the differences in lifetimes due to PKA phosphorylation were greatest with addition of myosin inhibitors: blebbistatin or vanadate. These results are consistent with the idea that cMyBP-C plays a role in stabilizing myosin on the thick filament and promoting the super-relaxed state, whereas its absence or phosphorylation leads to increased molecular disorder. For the cTnC probe, PKA treatment of myofibrils reduced the Ca2+-sensitivity of lifetime in both WT and cMyBP-C-/-. These results suggest unique phosphorylation-dependent roles for cMyBP-C and cardiac troponin I (cTnI) on thin filament structural dynamics. Our findings demonstrate that cMyBP-C and cTnI contribute to phosphorylation-dependent effects on thick and thin filament structural dynamics to influence force development. These TR-F probes can also be useful for screening of compounds that alter cMyBP-C interactions with the myofilaments.

Cardiac Myosin Binding Protein-C (MyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating heart failure. We hypothesize that PKA-mediated phosphorylation of human MyBP-C alters the structure of its N terminus, which leads to reduced myofilament binding and enhanced contractility. Mimicking the structural state of phosphorylated MyBP-C by small-molecule drugs could lead to therapy that optimizes MyBP-C function in the failing heart. Therefore, we have developed site-directed probes in MyBP-C capable of detecting structural changes due to phosphorylation using Time-Resolved FRET (TR-FRET). Site-directed mutagenesis of MyBP-C N-terminal domains C0 through C2 (C0-C2) was used to remove endogenous cysteines and introduce a pair of cysteines at strategic positions for labeling with donor and acceptor dyes. Replacement of native residues did not perturb binding to actin or myosin, verifying that the mutant C0-C2 was functional. TR-FRET results revealed that phosphorylation-mediated structural changes were most pronounced between a positively-charged loop in the C1 domain that interacts with tropomyosin and the tri-helix bundle region in the phosphorylatable M-domain, suggesting an important role for these regions in MyBP-C function. FRET efficiency changed by ∼17% between unphosphorylated and phosphorylated samples with a standard deviation of ∼0.5%. Fit to a Gaussian model, this corresponds to an increase in the distance between the two probes by ∼1.3 Å and an increase in the width of the distribution by ∼50%. The dynamic range and precision of the phosphorylation-mediated changes demonstrate excellent assay functionality for the purposes of high-throughput screening. These findings provide new molecular insight into the regulatory role of MyBP-C in contractility and demonstrate the potential of C0-C2 biosensors for structure-based screening of compounds in search of MyBP-C-targeted therapies for heart failure.

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.

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that modulates contractility by influencing interactions between myosin and actin. Despite being a leading cause of familial hypertrophic cardiomyopathy, the mechanism by which cMyBP-C modulates contractility is not well understood. N-terminal domains of cMyBP-C (C0 through C2) have been shown to interact with the actin-thin filament; however, the cMyBP-C regions involved in the interaction and how cMyBP-C influences actin's dynamic structure remain unclear. Here, we have used cosedimentation assays for measuring binding kinetics and time-resolved phosphorescence anisotropy (TPA) for measuring actin rotational dynamics. We examined the effects of actin interactions with human fragments C0-C1 and C0-C2 of cMyBP-C. We also evaluated roles of cMyBP-C phosphorylation sites known to influence contractility. We find that C0-C2 binds actin with ∼4-fold increase in apparent affinity compared to C0-C1. TPA of labeled-actin showed marked differences in the potency of effects between C0-C2, C0-C1, and phosphorylated cMyBP-C fragments with respect to decreasing the amplitude of torsional motions, increasing the rate of monomer rotation, and increasing relative resilience of the filament. A Hill equation was used to determine the level of cooperativity of the TPA effects for each fragment. Our results demonstrate that C0-C2 exhibits stronger cooperativity in propagating its effects on amplitude, rate, and resilience to more distant actin monomers than C0-C1. Assuming a thin filament cooperative unit of ∼7 actin monomers (length of tropomyosin), our TPA analyses suggest that one molecule of C0-C2 bound to actin propagated effects 2-3 cooperative units (14-21 monomers), whereas C0-C1 effects were confined to one unit, and phosphorylation reduced cooperativity. These findings provide new insight into the molecular basis for cMyBP-C effects on actin structure and function. This work was supported by an NIH R00HL122397 to B.A.C.

RATIONALE:Mutations in the gene encoding the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Mouse models targeting cMyBP-C and use of recombinant proteins have been effective in studying its roles in contractile function and disease. Surprisingly, while the N-terminus of cMyBP-C is important to regulate myofilament binding and contains many HCM mutations, an incorrect sequence, lacking the N-terminal 8 amino acids has been used in many studies.OBJECTIVES:To determine the N-terminal cMyBP-C sequences in ventricles and investigate the roles of species-specific differences in cMyBP-C on myofilament binding.METHODS AND RESULTS:We determined cMyBP-C sequences in mouse and human by inspecting available sequence databases. N-terminal differences were confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cosedimentation assays with actin or myosin were used to examine binding in mouse, human and chimeric fusion proteins of cMyBP-C. Time-resolved FRET (TR-FRET) with site-directed probes on cMyBP-C was employed to measure structural dynamics. LC-MS/MS supported the sequencing data that mouse cMyBP-C contains an eight-residue N-terminal extension (NTE) not found in human. Cosedimentation assays revealed that cardiac myosin binding was strongly influenced by the presence of the NTE, which reduced binding by 60%. 75% more human C0-C2 than mouse bound to myosin. Actin binding of mouse C0-C2 was not affected by the NTE. 50% more human C0-C2 than mouse bound to actin. TR-FRET indicates that the NTE did not significantly affect structural dynamics across domains C0 and C1.CONCLUSIONS:Our functional results are consistent with the idea that cardiac myosin binding of N-terminal cMyBP-C is reduced in the mouse protein due to the presence of the NTE, which is proposed to interfere with myosin regulatory light chain (RLC) binding. The NTE is a critical component of mouse cMyBP-C, and should be considered in extrapolation of studies to cMyBP-C and HCM mechanisms in human.

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.

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.