Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations

Abstract

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.

1. Introduction

Hypertrophic cardiomyopathy (HCM) is a heritable cardiac disease that affects one in 200–500 people [1]. HCM is characterized by a hypercontractile phenotype, resulting in diastolic dysfunction and cardiac remodeling, which can lead to heart failure (HF) [2]. A leading cause of HCM is mutations in MYBPC3 (the gene encoding cMyBP-C), comprising ~40–60% of all HCM associated mutations [3]. cMyBP-C is a sarcomeric protein that interacts with both myosin and actin to facilitate normal contraction and relaxation of the heart [4,5]. Phosphorylation of cMyBP-C fine-tunes cardiac function by regulating its interactions with myosin and actin [4,6]. While cMyBP-C is highly phosphorylated under healthy conditions, in disease, including those associated with mutations in other sarcomeric proteins, phosphorylation levels of cMyBP-C are significantly reduced [7]. Modified protein interactions can arise from mutations in sarcomeric proteins. How sarcomeric point mutations trigger contractile dysfunction and lead to the variable disease presentation (symptoms, severity, progression, and remodeling) in HCM are incompletely understood, and this significantly impedes therapeutic development. Understanding how normal and HCM-associated cMyBP-C variants are affected by phosphorylation-sensitive binding to myosin and actin will aid in this understanding of cMyBP-C in normal physiology, HCM disease, and as a target for new medicines [8].

cMyBP-C is a thick filament-associated sarcomeric protein comprised of 8 immunoglobulin-like and 3 fibronectin-like domains termed C0-C10 (Fig. 1 A and D). In myocardium, the C-terminus (domains C8-C10) is anchored to the thick filament backbone with binding sites to light meromyosin [9]. The N-terminal domains (domains C0 through C2, i.e., C0-C2) contain binding sites for myosin and actin [10]. In the C0-C2 fragment, C0 and C1 domains are connected by a proline-alanine-rich linker (P/A) and C1 and C2 are connected by the M-domain. The M-domain has a largely disordered N-terminal portion containing four serine residues that can be phosphorylated: S275, S284, S304, S311 and an ordered C-terminal subdomain comprised of 3 connected α-helical segments arranged in a tri-helix bundle (THB) [11].

Fig. 1.

Organization of the sarcomere and the positioning of the FRET probes on myosin, actin, and cMyBP-C. (A) Cartoon of the sarcomere showing the actin-containing thin filaments, myosin-containing thick filaments, and titin between two Z-discs. The C-zone, the thick filament region containing cMyBP-C molecules (blue vertical stripes), is indicated. The N-terminal domains (C0-C2) of cMyBP-C are circled in the space between the thin and thick filaments. (B) Subunits and domains of β-cardiac myosin showing two heavy chains (grey), two essential light chains (ELC) and two regulatory light chains (RLC) are depicted. FMAL labeling of RLC is indicated by the yellow triangle. A ribbon diagram of the crystal structure of RLC (PDB: 5TBY) is shown (purple) with the position of amino acid 105 used for FMAL labeling indicated (yellow triangle). (C) F-actin consisting of G-actin monomers (green balls) with FMAL labeling indicated by yellow triangles. A ribbon diagram of the crystal structure of monomeric actin (PDB: 3HBT) is shown (green) with the position of amino acid 374 used for FMAL labeling indicated. (D) Cartoon of cMyBP-C. Immunoglobulin-like domains (circles) and fibronectin type-III domains (hexagons) are depicted. The proline/alanine rich linker (P/A) and M-domain (M) that contains phosphorylation sites (P) and tri-helix bundle (THB) are shown. (E) N-terminal C0-C2 used in this study is shown with acceptor probe sites indicated (fuchsia triangles). (F–H) Ribbon diagrams are shown of C0 (PDB: 2K1M) and C1 (PDB: 2V6H) domains, and the THB (PDB: 5K6P) with the sites used for TMR labeling indicated (fuchsia triangles). Created, in part, with Biorender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Binding of N-terminal cMyBP-C to actin and myosin is strongest in the unphosphorylated state. This binding can activate the thin filament through interactions with actin and tropomyosin, and separately act as a brake on contraction by impeding activation of the thick filament [12–14]. cMyBP-C binding can slow relaxation of the heart through postulated drag mechanisms and sequester myosin heads to favor an inhibited super-relaxed state (SRX) of myosin [5,15]. The SRX describes decreased myosin ATPase activity, associated with the folded back interacting-heads motif (IHM) structure of myosin, which could slow cross-bridge formation and force development [16]. Downstream activation of the β-adrenergic signaling pathway leads to phosphorylation of sarcomeric proteins, including cMyBP-C, for proper contraction and relaxation of the heart under varying physiological demands. This includes relieving the constraint on myosin IHM structure and/or SRX function through varying phosphorylation levels of cMyBP-C [17]. Phosphorylation of cMyBP-C mediated by protein kinase A (PKA), protein kinase C (PKC) and Ca²⁺-calmodulin-activated kinase II (CaMKII) results in structural changes [18–20], altered interactions with myosin and actin [4,21], and/or changes in these activating and braking mechanisms [4].

Phosphorylation of cMyBP-C may be a critical mediator of diastole by normally enhancing relaxation, and reduced phosphorylation in cardiac disease (resulting in increased binding to myosin and actin) may cause diastolic dysfunction [22]. Indeed, cMyBP-C phosphorylation is commonly dysregulated and reduced in patients with HCM or HF [23], which could perturb normal cMyBP-C interactions with myosin and actin in contraction and relaxation. Phosphorylated cMyBP-C may be cardioprotective as mice expressing phosphomimetic charge substitutions at PKA-mediated phosphorylation sites display preserved cardiac morphology and resistance to ischemic-reperfusion injury [24], enhanced myocardial relaxation [22], and attenuated age-related cardiac dysfunction [25]. That cMyBP-C phosphorylation is reduced in HCM and HF suggests that modified binding of cMyBP-C to actin and/or myosin may contribute to the disease pathology by affecting contractile properties of the myocardium [4,26]. cMyBP-C could play a role in systole, as well as diastole, by sustaining force and stiffness to extend through the entirety of the cardiac ejection period [27].

The first FDA-approved drug for patients with obstructive HCM, mavacamten, is a cardiac myosin inhibitor that was successful in improving symptoms and cardiac function [28,29]. Mutations in HCM have been linked to destabilization of the energy-conserving SRX of myosin [30]. Thus, the effectiveness of mavacamten to mitigate hypercontractility in obstructive HCM patients has been associated with stabilizing the SRX [31]. This advancement in treating HCM by a small-molecule drug has led to increased efforts in searching for and developing drugs that target myosin as well as other sarcomeric proteins. Compounds binding to cMyBP-C that mimic phosphorylated cMyBP-C and reduce its myosin or actin binding to influence contractility may therefore represent useful treatments for HCM and HF.

Hundreds of mutations in MYBPC3 (the gene encoding cMyBP-C) are linked to the development of cardiomyopathy and progression to HF [32,33], but the impact of these numerous mutations of cMyBP-C and its phosphorylation on interactions with myosin and actin remains limited, in part, due to the lack of scalable, high-throughput assays of protein function. We recently developed high-throughput fluorescence lifetime (FLT)-based assays to study the dynamic structure of C0-C2 [19] and the interactions between C0-C2 and actin as affected by phosphorylation and HCM-associated variants of cMyBP-C [34]. Screening of 1280 pharmacologically active compounds with one of these assays identified the first 3 drugs that bind to cMyBP-C and abolish its interactions with actin [35]. These drugs were further characterized for C0-C2-actin binding using a FLT-fluorescence resonance energy transfer (FRET)-based C0-C2-actin binding assay [35]. In this assay, a donor fluorescent probe (fluorescein-5-maleimide (FMAL)) was attached to actin on cys374 (Fig. 1C) and an acceptor probe (tetramethylrhodamine (TMR)) was attached to the C1 domain on cys249 in C0-C2 (Fig. 1G). Binding of C0-C2 to actin brought the acceptor probe in close proximity to the donor probe resulting in FRET. FLT-FRET was detected as a change in donor-acceptor lifetime (the time it takes for the donor intensity to decay from its maximum intensity following excitation, to ~37% (1/e), with acceptor(s) nearby). We then used the C0-C2-actin FLT-FRET assay (as well as monitoring TMR-C0-C2 FLT as a readout of binding) to screen a 2684 compound library of FDA-approved drugs and active pharmaceutical ingredients and identified 2 other drugs that affect myosin ATPase in cardiac myofibrils [36].

Here, we further developed the C0-C2-actin FLT-FRET assays and, for the first time, established an assay to accurately measure interactions between cMyBP-C and myosin. For myosin labeling, the donor, FMAL, was placed on the RLC subunit of myosin containing the cys105 mutation (Fig. 1B). RLC has no endogenous cysteine residues and introduction of a cysteine at residue 105 (cys105) provides a convenient site for thiol labeling [37]. This was then exchanged onto purified full-length cardiac myosin [38]. C0-C2 containing a single cysteine in the C0 (cys85), C1 (cys249), or THB of the M (cys330) domains was used for acceptor (TMR) labeling (Fig. 1E–1H). The FMAL-TMR FRET pair has an R₀ of 5.5 nm, implying that probes separated by up to 8.25 nm (~1.5 × R₀) can be reliably detected, which is an appropriate distance for expected C0-C2-myosin interactions modeled from computer docking [39].

Using these FLT-FRET assays, we first demonstrated the sensitivity of the assays for M-domain phosphorylation and then examined the effects of 5 N-terminal cMyBP-C mutations (T59A, R282W, E334K, L349R, and L352P) that are causative for HCM or have predictable effects on myosin and actin interactions. Four of the 5 mutations we selected are located in M-domain and are expected to increase or decrease actin and/or myosin binding. One mutation (R282W) is located in the PKA recognition sequence of a phosphorylation site (S284), and 3 others (E334K, L349R, and L352P) are in the THB region of M-domain. The fifth mutation selected is located in domain C0, which we expected to have little to no effect on binding due to T59 being a non-conserved residue. While each of these mutations have been previously studied [21,34,40–44], here we report both myosin and actin binding effects in complementary FLT-FRET assays in similar experimental conditions. We investigated how N-terminal cMyBP-C interacts with myosin and actin and changes with HCM-associated mutations with and without phosphorylation. Lastly, we examined the potential usefulness of these FLT-FRET assays for drug library screening purposes to identify small-molecule compounds that modulate C0-C2 interactions with myosin and actin with high precision.

2. Materials and methods

2.1. Myosin filament preparations

Cardiac myosin from porcine ventricles (Pel-Freez Biologicals) was prepared as described by Rohde et al. [45] and further purified with ion-exchange chromatography (HiPrep DEAE FF 16/10 column, GE Healthcare) to remove contamination of thin filament proteins and cMyBP-C. The column was equilibrated with, and myosin was applied in, 40 mM sodium pyrophosphate at pH 7.5. Myosin was eluted with a gradient of 0–500 mM KCl in 20 mM sodium pyrophosphate at pH 7.5, over 100 ml at 2 ml/min, and collected in 5 ml fractions (AKTA Prime Plus, GE Healthcare). SDS-PAGE was used to determine the fractions to pool with <2% of actin contamination. The purified myosin was dialyzed into 600 mM KCl, 25 mM KP_i, 2 mM DTT, pH 7.0 buffer. For storage at −80 °C, sucrose was added to 150 mM (final concentration), and flash frozen in a dropwise manner into liquid nitrogen.

2.2. RLC preparation and labeling

Human RLC (from the myosin light chain 2 gene, MYL2) coding sequence with E. coli optimized codons was inserted into the pET45b expression vector (GenScript). Mutagenesis of cys105 from valine to cysteine was performed using a Q5 Site-Directed Mutagenesis Kit (New England Bio Labs) and confirmed by DNA sequencing (Eton Biosciences). For protein production, the RLC expression plasmid was transformed into E. coli BL21DE3-competent cells (New England Bio Labs) and grown at 37 °C in Overnight Express Autoinduction medium (Novagen) supplemented with 100 μg/ml ampicillin. RLC was purified by inclusion body isolation followed by ion exchange chromatography. Bacteria were harvested by centrifugation for 5 min, 4 °C, 8000 × g, resuspended in phosphate buffered saline (154 mM NaCl, 0.8 mM KH₂PO₄, 5.6 mM Na₂HPO₄, pH 7.4) (5–10 ml/g cell paste) containing 0.1 mM PMSF, and again collected by centrifugation for 20 min, 4 °C, 3300 × g, and frozen at −80 °C until use. The frozen bacterial cell pellet was resuspended in lysis buffer (25 mM Tris-Cl, 5 mM EDTA, 1 mM PMSF, 50 mM glucose, pH 8.0) and disrupted using an Emulsiflex homogenizer (Avestin). MgCl₂ was added (to a final concentration of 10 mM) followed by the addition of DNAse 1 (to a final concentration of 5 mg/ml) and incubation at 4 °C for 1 h with rocking. Triton X-100 was added (from a 10% stock) to a final concentration of 0.1%. To collect insoluble proteins, including RLC in inclusion bodies, the lysed bacteria were centrifuged for 15 min, 4 °C, 20,000 × g in a Beckman JA-17 rotor. The inclusion bodies were washed 2 × by resuspension in lysis buffer plus Triton X-100 (0.1%) followed by centrifugation and finally washed with lysis buffer without Triton X-100. The resulting pellet was frozen at −20 °C until further purification. RLC inclusion bodies collected from 1 L of bacterial culture were resuspended in 20 ml resuspension buffer (7 M Urea, 30 mM Tris-HCl, 50 mM NaCl, pH 7.5, and 1 mM DTT (final concentration added just prior to use)). This was stirred at room temperature (23 °C) for 30 min or until the pellet was completely dissolved. Any remaining insoluble debris was removed by centrifugation (30 min, 4 °C, 30,000 × g in a Beckman JA-17 rotor). The supernatant containing denatured RLC was applied to an ion exchange column (Hitrap Q XL, GE Healthcare). The column was first equilibrated with 4 M Urea, 30 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, pH 7.5. RLC was eluted in a gradient of 50–250 mM NaCl (and 4 M Urea, 30 mM Tris-HCl, 1 mM DTT, pH 7.5) over 50 ml at 1 ml/min, collecting 1 ml fractions (AKTA Prime Plus, GE Healthcare). Fractions were analyzed by SDS-PAGE and those containing pure RLC were pooled and dialyzed into 0.4 mM KCl. The concentration was adjusted to 250 mM and, flash frozen in liquid nitrogen and stored at −80 °C until use.

Labeling of 50 μM RLC with fluorescein-5-maleimide (FMAL, Invitrogen) was done in TUKE buffer (50 mM Tris, 6 M Urea, 80 mM KCl, 1 mM EDTA, pH 7.5). Since frozen RLC was 250 mM in 0.4 mM KCl, it was diluted 5 × with 1.25 × TUKE buffer lacking KCl (62.5 mM Tris, 7.5 M Urea, 1.25 mM EDTA, pH 7.5). TCEP was added (200 μM final concentration) and incubated with the RLC for 30 min at 23 °C, followed by the addition of FMAL (from a 20 mM stock in DMF stored at −80 °C) to a final concentration of 275 μM. Labeling was done for 2 h at 23 °C and terminated by the addition DTT (to a final concentration of 1.5 μM). Unincorporated dye was removed by extensive dialysis into exchange buffer (50 mM NaCl, 5 mM EDTA, 2 mM EGTA, 10 mM NaPO₄, pH 7.5). Following dialysis, labeled RLC was centrifuged (30 min, 4 °C, 100,000 RPM (350,000 × g)) in a Beckman TLA-120.2 rotor to remove any precipitated dye and insoluble protein. The degree of labeling, typically 60–80%, was determined by using FMAL’s extinction coefficient (E₄₉₄ = 68,000 cm⁻¹ M⁻¹) and the concentration of RLC was determined by using a Pierce^™ BCA assay (Thermo Fisher Scientific) with BSA as a standard. FLT-FRET between the RLC of myosin and C0-C2 was dependent on RLC’s localization to the myosin heavy chain, as 1 μM of unincorporated RLC under the same conditions resulted in <2% FRET efficiency (Fig. S1).

2.3. RLC exchange onto myosin

RLC exchange onto myosin was performed as described [38], adjusted for the use of full-length myosin. Labeled RLC and purified myosin, in exchange buffer containing 1 mM DTT, were mixed to final concentrations of 25 μM of 10% labeled RLC (2.5 μM FMAL labeled RLC, 22.5 μM unlabeled RLC) and 5 μM of myosin. ATP was added to a final concentration of 2 mM. After a 50 min incubation at 42 °C, MgCl₂ (from a 500 mM stock) was slowly added to 15 mM to stop the exchange. To remove excess RLC, the mixture was centrifuged for 10 min at 4 °C, at 200,000 × g in a Beckman TLA-120.2 rotor, pelleting the myosin filaments. The supernatant containing excess RLC was removed, and the pellet was washed with exchange buffer containing 15 mM MgCl₂. The pellet was centrifuged at 200,000 × g for an additional 5 min in the wash solution. The supernatant was removed, and the final pellet was resuspended in 600 mM NaCl, 0.2 mM EDTA, 25 mM Tris, pH 7.0, clarified by centrifugation at 4 °C, 15,000 RPM (21,000 × g) for 10 min in an Eppendorf 5424R benchtop microfuge and then dialyzed into 75–20–3 buffer (75 mM KCl, 20 mM Tris, 3 mM MgCl₂, 250 μM ATP, 1 mM DTT, pH 7.0) for FLT-FRET experiments.

2.4. Actin filament preparations and labeling

Actin was prepared from rabbit skeletal muscle by extracting acetone powder in cold water as described [34,46]. Actin was labeled as F-actin. 50 μM G-actin (in 10 mM Tris, 0.2 mM CaCl₂, 0.2 mM ATP, pH 7.5) was brought to 20 mM Tris (pH 7.5) and then polymerized by the addition of MgCl₂ to a final concentration of 2 mM and KCl to a final concentration of 100 mM. Polymerization was allowed to proceed for 1 h at 23 °C. FMAL was added to a final concentration of 0.5 mM (from a 20 mM stock in DMF stored at −80 °C). Labeling was done for 1 h at 23 °C and stopped by the addition of DTT to 2.5 mM. Labeled F-actin was collected by centrifugation (30 min, 100,000 RPM (350,000 × g) in a Beckman TLA-120.2 rotor at 4 °C). The F-actin pellet was rinsed 3 times with actin labeling G-buffer (5 mM Tris, 0.2 mM CaCl₂, 0.5 mM ATP, pH 7.5) containing 3 mM MgCl₂ and then once with actin labeling G-buffer and then resuspended in actin labeling G-buffer. Labeled G-actin was clarified to remove any remaining F-actin or precipitated FMAL by centrifugation (10 min, 90,000 RPM in a Beckman TLA-120.2 rotor at 4 °C). The extent of labeling (typically ~70%) was determined by UV–vis spectroscopy. Labeled G-actin was mixed with unlabeled G-actin to achieve a 10% labeled mixture. This was polymerized by the addition of MgCl₂ to a final concentration of 2 mM and KCl to a final concentration of 100 mM, and then dialyzed against MOPS actin-binding buffer (M-ABB; 100 mM KCl, 10 mM MOPS, pH 6.8, 2 mM MgCl₂, 0.2 mM CaCl₂, 0.2 mM ATP, 1 mM DTT, 1 mM sodium azide). Any bundled actin was removed by centrifugation at 4 °C, 15,000 RPM (21,000 × g) for 10 min in an Eppendorf 5424R benchtop microfuge. Actin concentration and % FMAL labeling was again determined by UV–vis spectroscopy. The labeled F-actin was stabilized with the addition of phalloidin (to an equal molar concentration as actin).

2.5. Recombinant human cMyBP-C and labeling

pET45b vectors encoding E. coli optimized codons for the C0-C2 portion of human cMyBP-C with N-terminal 6 × His tag and TEV protease cleavage site were obtained from GenScript. C0-C2 mutants were engineered using a Q5 Site-Directed Mutagenesis Kit (New England Bio Labs). Substitution mutations were performed to generate C0-C2 constructs containing a single cysteine located in the C0-domain at position 85, C1-domain at position 249, or the THB in the M-domain at position 330 (termed C0-C2^cys85, C0-C2^cys249, and C0-C2^cys330). C0-C2 lacking endogenous cysteines and C0-C2^cys249 have been described [19]. To insert cysteines of interest for probe labeling, mutation S85C (C0-C2^cys85) and P330C (C0-C2^cys330) were added to C0-C2 lacking endogenous cysteines. cMyBP-C mutations T59A, R282W, E334K, L349R, and L352P, were introduced to each C0-C2^cys85, C0-C2^cys249, and C0-C2^cys330 construct. This resulted in C0-C2 constructs containing a single cysteine in the C0, C1, or M-domain with a cMyBP-C mutation inserted: C0-C2^{cys85, T59A}, C0-C2^{cys85, R282W}, C0-C2 ^{cys85, E334K}, C0-C2^{cys85, L349R}, C0-C2^{cys85, L352P}, C0-C2^{cys249, T59A}, C0-C2^{cys249, R282W}, C0-C2^{cys249, E334K}, C0-C2^{cys249, L349R}, C0-C2^{cys249, L352P}, C0-C2^{cys330, T59A}, C0-C2^{cys330, R282W}, C0-C2^{cys330, E334K}, C0-C2^{cys330, L349R}, C0-C2^{cys330, L352P}. All sequences were confirmed by DNA sequencing (Eton Biosciences). Protein production in E. coli BL21DE3-competent cells (New England Bio Labs) and purification of C0-C2 protein using His60 Ni Superflow resin (Thermo Fisher Scientific) were done as described [34]. C0-C2 (with His-tag removed by TEV protease digestion) was further purified using size-exclusion chromatography to achieve >90% intact C0-C2 as described [13] and then concentrated, dialyzed to 50/50 buffer (50 mM NaCl and 50 mM Tris, pH 6.7), and stored at 4 °C. For long term storage at −20 °C, glycerol, containing 50 mM NaCl and 50 mM Tris, pH 6.7, was added to 50% (v/v).

C0-C2 was labeled with tetramethylrhodamine-5-maleimide (TMR, AnaSpec. Inc.) in 50/50 buffer. C0-C2 (50 μM) was first treated with TCEP (200 μM) for 30 min at 23 °C while rocking. TMR (stored as a 20 mM stock in DMF at −80 °C) was added to final concentrations of 75 μM (C0-C2^cys85 constructs), 125 μM (C0-C2^cys249 constructs), or 140 μM (C0-C2^cys330 constructs). Labeling was done for 1 h at 23 °C and terminated by the addition of 5-fold molar excess of DTT. Unincorporated dye was removed by extensive dialysis against 75–20–3 buffer (for myosin binding) or M-ABB buffer (for actin binding). Following the dialysis, labeled C0-C2 was centrifuged for 30 min, 4 °C at 100,000 RPM (350,000 × g) in a Beckman TLA-120.2 rotor to remove any precipitated dye and insoluble protein. The degree of labeling ranged from 50 to 90% dye/C0-C2 as measured by absorbance. To attain uniform 50–60% dye/C0-C2 for FLT-FRET experiments, unlabeled corresponding C0-C2 construct was added.

2.6. Protein and dye concentration

A BCA Assay and SDS-PAGE analysis using a BSA protein standard were used to determine protein concentrations. The extinction coefficient for FMAL is 68,000 cm⁻¹ M⁻¹ at 494 nm and the extinction coefficient for TMR is 91,000 cm⁻¹ M⁻¹ at 542 nm, provided in the manufacturer’s specifications.

2.7. PKA preparation and in vitro phosphorylation of cMyBP-C

PKA catalytic subunit α was obtained from Addgene (Plasmid #14921), expressed in E. coli BL21DE3 competent cells (New England Bio Labs), and grown at 23 °C in Overnight Express Autoinduction medium (Novagen) supplemented with 100 μg/ml carbenicillin. PKA was purified using His60 Ni Superflow resin (Thermo Fisher Scientific) as described for C0-C2 [34]. PKA was eluted from the resin in Elution buffer (50 mM Na₂HPO₄, 200 mM NaCl, 250 mM imidazole, pH 8.0) and then dialyzed into storage buffer (25 mM KPO₄, 150 mM NaCl, 1 mM TCEP (tris(2-carboxyethyl)phosphine), pH 7.0). PKA was concentrated to 2 mg/ml and mixed with an equal volume of storage buffer containing 100% glycerol (v/v). PKA was aliquoted, flash frozen in liquid nitrogen, and stored at −80 °C until use. After aliquots were removed from −80 °C they were stored at −20 °C and used within 2 weeks. C0-C2 was treated with 7.5 ng PKA/μg C0-C2 at 30 °C for 30 min, which is 3 times the level (2.5 ng PKA/μg C0-C2) needed to achieve maximal phosphorylation as determined by in-gel staining of proteins with Pro-Q Diamond (Thermo Fisher Scientific) [13,34].

2.8. Steady-state basal and actin-activated ATPase activity of myosin

An NADH-coupled assay was used to measure the steady-state ATPase activity of myosin. The assay was performed at 23 °C in buffer containing 10 mM KCl, 4 mM MgCl₂, 20 mM Tris-HCl, pH 7.5. Each reaction contained 1 μM of myosin ±4 μM of actin and 2 mM phosphoenolpyruvate, 0.3 mM NADH, 38 U/ml pyruvate kinase, 50 U/ml lactate dehydrogenase, and 2 mM MgATP. Change in NADH absorption at 340 nm was measured over 15 min using a Beckman DU730 UV–Vis spectrophotometer. The rates, ADP myosin⁻¹ s⁻¹, (commonly expressed as s⁻¹) were derived from this data [47].

2.9. Fluorescence lifetime-based FRET (FLT-FRET) data acquisition and analysis

Fluorescence lifetime measurements were acquired using a high-precision fluorescence lifetime plate reader (FLTPR; Fluorescence Innovations, Inc.) [19,34]. For FLT-FRET experiments, FMAL was excited with a 473-nm microchip laser (Bright Solutions) and emission was filtered with 488-nm long-pass and 517/20-nm band-pass filters (Semrock). The photomultiplier tube (PMT) voltage was adjusted so that the peak signals of the instrument response function (IRF) and the biosensor were similar (~100 mV). The observed time-resolved fluorescence waveforms for each well were fit by convolving the IRF with a single-exponential decay to determine the FLT $\left(\mathrm{\tau }\right)$ of the excited fluorophore (Eq. 1) [19,34]. The decay of the excited state of the fluorescence FMAL dye attached to myosin or actin to the ground state is:

$$\mathrm{I}(\mathrm{t})=\mathrm{F}(\mathrm{t}{)}^{\mathrm{*}}\mathrm{I}\mathrm{R}\mathrm{F}$$

$$\mathrm{F}\left(t\right)=I_{0}exp(-t/\tau )$$ (1)

Where I is the measured waveform, $I_0$ is the fluorescence intensity upon peak excitation ($t=0$), and $\mathrm{\tau }$ is the FLT ($t=\mathrm{\tau }$ when $I$ decays to $1/e$ or ~37% of $I_0$). The efficiency of energy transfer E (FRET efficiency) was calculated as:

$$\mathrm{E}=1-{\mathrm{\tau }}_{\mathrm{D}\mathrm{A}}/{\mathrm{\tau }}_{\mathrm{D}}$$ (2)

Where ${\mathrm{\tau }}_{\mathrm{D}\mathrm{A}}$ is the lifetime of FMAL-myosin or FMAL-actin in the presence of TMR-C0-C2 and ${\mathrm{\tau }}_{\mathrm{D}}$ is the average lifetime of FMAL-myosin or FMAL-actin in the absence of C0-C2. Fluorescence waveforms of FMAL-labeled myosin and actin, in the absence and presence of TMR-labeled C0-C2 were analyzed by one-exponential fitting to determine fluorescence lifetime (Fig. S2).

2.10. Determination of FLT-FRET_max and EC₅₀ values

The maximum FLT-FRET (FLT-FRET_max) and EC₅₀ values for C0-C2 binding to myosin and actin were determined by fitting the data to a quadratic model (Michaelis-Menten function) using Origin Pro 2023 computer software package through a nonlinear least-squares minimization (Levenberg-Marquardt algorithm) as previously described [34,46]. These EC₅₀ and FLT-FRET_max values are used as comparative indicators of binding characteristics for C0-C2 binding to myosin and actin under different conditions (±phosphorylation or in the presence of mutations). The adjusted R² was used to test the goodness-of-fit to the model.

2.11. Determination of $Z^{\prime }$-factor for C0-C2^cys249

For suitability in high-throughput screening (HTS), the lifetime of FMAL attached to myosin or actin was determined in multiple wells in the absence or presence of 5 μM TMR-labeled C0-C2^cys249. The FLT assay quality was determined by computing $Z^{\prime }$ in the absence $\left({\mathrm{\tau }}_{\mathrm{A}}\right)$ and presence of TMR-C0-C2^{cys249 $\left({\mathrm{\tau }}_{\mathrm{B}}\right)$}:

$${\mathrm{Z}}^{\prime }=1-3\left(\frac{{\sigma }_{\mathrm{A}}+{\sigma }_{\mathrm{B}}}{\left|{\mu }_{\mathrm{A}}-{\mu }_{\mathrm{B}}\right|}\right)$$ (3)

where ${\sigma }_{\mathrm{A}}$ and ${\sigma }_{\mathrm{B}}$ are the standard deviations (SD) of the ${\mathrm{\tau }}_{\mathrm{A}}$ and ${\mathrm{\tau }}_{\mathrm{B}}$ lifetimes, respectively, ${\mu }_A$ and ${\mu }_B$ are the means of the ${\mathrm{\tau }}_{\mathrm{A}}$ and ${\mathrm{\tau }}_{\mathrm{B}}$ lifetimes, respectively. A $Z^{\prime }$ value of <0 is indicative of “useless” conditions, whereas 0 to 0.5 is “good” and 0.5 to 1.0 is “excellent” assay quality for use in HTS [48].

2.12. Statistics

Average data are provided as mean ± standard error (SE). Each experiment was done with >2 separate protein preparations. Statistical significance (p-value) is denoted in each figure and is evaluated by Student’s t-test, 1-way ANOVA or 2-way ANOVA with post-hoc analysis. Biological repeats (N, independent protein preparations) and technical repeats (n, independent reactions) for each individual data point are indicated in figure and table legends.

3. Results

3.1. FRET probes for detection of C0-C2 binding to myosin and actin

Site-directed mutagenesis was used to introduce a cysteine in the RLC subunit at residue 105 (Fig. 1B). To ensure that the FMAL on one RLC did not interact with FMAL on a neighboring RLC (i.e., inter-RLC FRET), we exchanged only 10% labeled RLC onto myosin [38]. RLC exchange was monitored by analyzing SDS-PAGE gels for ratios of protein and fluorescence staining (Fig. 2A). Similarly, F-actin was labeled to 10% at cys374 (Fig. 1C) to reduce effects of FMAL on neighboring actin monomers (i.e., inter-actin FRET). To monitor C0-C2-myosin and C0-C2-actin binding, we inserted acceptor probes at different positions (C0: cys85; C1: cys249; the THB in the M-domain: cys330) in C0-C2 (Fig. 1E–G).

Fig. 2.

FMAL-labeled RLC exchange onto myosin and ATPase activity. (A) Representative SDS-PAGE gel monitoring steps of the Myosin-RLC exchange protocol. Lane 1, Protein molecular weight markers (MWM); lane 2, unlabeled RLC; lane 3, FMAL-labeled RLC; lane 4, cardiac myosin; lane 5, mixture of myosin with 5-fold excess FMAL-labeled RLC; lanes 6 and 7, washes removing excess RLC; lane 8, final myosin containing FMAL-labeled RLC. The top image is the Coomassie stained gel, and the bottom gel section shows fluorescence signal of labeled RLC for each lane. See Materials and Methods for further details. (B) ATPase activity of 1 μM of myosin without RLC exchange (0.040 ± 0.005 s⁻¹) in solid circles and RLC-exchanged myosin (0.07 ± 0.003 s⁻¹) in open circles were similar in the absence of actin (left) and for actin-activated ATPase activity (0.167 ± 0.029 s⁻¹ for unexchanged myosin and 0.161 ± 0.012 s⁻¹ for RLC-exchanged myosin) (right). Data is shown as mean ± SE for N ≥ 3 independent biological replicates (protein preparations) and n ≥ 4 technical repeats (wells of binding reactions). For comparisons between unexchanged and RLC-exchanged myosin, no significant differences were observed, and actin stimulation was significant for both (p < 0.001, 2-way ANOVA with Tukey’s post-hoc test.

To test FMAL-RLC-exchanged myosin function, we measured the steady-state ATPase rates and compared them to unexchanged myosin (Fig. 2B). The basal and actin-activated myosin ATPase activities of 0.040 ± 0.005 s⁻¹ and 0.167 ± 0.029 s⁻¹ for the RLC-exchanged myosin were comparable to the rates of unexchanged myosin with ATPase activities of 0.047 ± 0.003 s⁻¹ and 0.161 ± 0.012 s⁻¹, respectively, demonstrating that the labeled myosin used for FLT-FRET experiments was functional.

3.2. C0-C2-myosin and C0-C2-actin FLT-FRET binding curves

Binding curves were generated by calculating the FRET efficiency resulting from incubating 1 μM FMAL-labeled myosin or actin with increasing concentrations of TMR-C0-C2 (with the acceptor probes on cys85, cys249, or cys330) (Fig. 3). PKA phosphorylation of TMR-C0-C2 reduced FRET efficiency with both myosin and actin binding (Fig. 3). Binding parameters (EC₅₀, maximum FRET efficiency (FLT-FRET_max), and adjusted R² (adj. R²)) for the curves in Fig. 3 can be found in Table S1. The C0-C2-actin binding curves, both unphosphorylated and phosphorylated (Fig. 3D–F), fit well to a quadratic model (adj. R²s of 0.99–1.00). Unphosphorylated C0-C2 binding to myosin (Fig. 3A–C) fit well to the model (adj. R² = 0.93–0.96), but the phosphorylated C0-C2-myosin binding did not (adj. R² = 0.60–0.88).

Fig. 3.

Myosin-C0-C2 and actin-C0-C2 FLT-FRET binding curves. FLT-FRET resulting from the binding of unphosphorylated (solid lines) and phosphorylated (dashed lines) TMR-C0-C2 to FMAL-myosin (A–C) and FMAL-actin (D–F). Panels A and D: TMR FRET acceptor is on C0, C0-C2^cys85. Panels B and E: TMR FRET acceptor is on C1, C0-C2^cys249. Panels C and F: TMR FRET acceptor is in the THB C0-C2^cys330. EC₅₀, maximal FRET (FLT-FRET_max), and adjusted R² are shown in Table S1. Myosin binding with C0-C2 + PKA data points displayed low R² values (Table S1) and were therefore not fit to the quadratic model (A–C). Data are provided as mean ± SE for N ≥ 3 independent biological replicates (protein preparations) and n ≥ 10 technical repeats (wells of binding reactions). These actin-C0-C2 FLT-FRET binding curves are compared with cosedimentation curves in Fig. S3. Arrows indicate FLT-FRET values from 5 μM TMR-C0-C2^cys249 used in $Z^{\prime }$ calculations (Table S3). Arrowheads in all graphs indicate the concentration (2.5 μM) used for comparison of mutant binding (Table 1).

For actin, we determined the similarities between the FLT-FRET binding curves to cosedimentation assays using FMAL-labeled actin and TMR-C0-C2 (with the acceptor probes on cys85, cys249, and cys330) by superimposing binding curves and normalized correlation plots (Fig. S3 and S4).

3.3. High-throughput screening (HTS) potential of C0-C2-myosin and C0-C2-actin FLT-FRET binding assays

To test the suitability of the myosin and actin FLT-FRET binding assays in HTS for compounds that modify C0-C2 binding, the average and standard deviation (SD) values for the lifetimes of FMAL-myosin and FMAL-actin were measured in the absence and presence of 5 μM TMR-C0-C2^cys249 (see arrows in Fig. 3B and E) and used to calculate a $Z^{\prime }$ factor (see Materials and Methods). Results from 4 independent preparations yielded $Z^{\prime }$ ranges of 0.76–0.90 for myosin and 0.94–0.95 for actin, indicating that these are “excellent” conditions for use in HTS to detect compounds that increase or decrease cMyBP-C FRET (binding) with myosin or actin (Table S3).

3.4. Effects of C0-C2 mutations associated with HCM on FLT-FRET with myosin and actin

FLT-FRET assays were used to assess effects of 5 mutations in N-terminal cMyBP-C on phosphorylation-sensitive cMyBP-C binding to myosin or actin: T59A in C0, R282W in a PKA phosphorylation target recognition sequence, and E334K, L349R, and L352P in the THB of the M-domain (Fig. 4). These five mutations were introduced into C0-C2 proteins that contained each of the 3 acceptor probe sites. These 15 TMR-labeled mutants and 3 WT TMR-C0-C2 proteins were combined with FMAL-labeled myosin or actin and the resulting FLT-FRET was measured. Table 1 shows the relative FRET efficiency resulting from 2.5 μM TMR-C0-C2 for each mutation, unphosphorylated and phosphorylated, interacting with myosin or actin compared to that of WT TMR-C0-C2 controls. The concentration, 2.5 μM TMR-C0-C2, shown in this table shows a submaximal level of binding (Fig. 3, arrowheads). At this concentration, FLT-FRET has the capacity to detect increases and decreases in binding and is therefore a useful assay condition to survey increases or decreases in binding due to the mutations. FRET efficiencies (%, see Materials and Methods) observed between labeled myosin and 2.5, 5 and 10 μM TMR-C0-C2 are presented in Fig. S5–S7. Complete binding curves for actin with their respective EC₅₀ and FLT-FRET_max values are found in Fig. S8 and Table S4.

Fig. 4.

cMyBP-C C0-C2 organization and M-domain mutants. (A) Cartoon depicting C0-C2. The P/A-rich linker (P/A) and M-domain (M), containing PKA phosphorylation sites (P) and tri-helix bundle (THB), are indicated. (B) The sequence of M-domain and locations of mutations (red, R282W, E334K, L349R, and L352P) tested for effects on myosin and actin binding is shown. Serine residues phosphorylated by PKA (green), the PKA recognition sequences (highlighted in yellow), and helix residues in the THB (thick underlines) are indicated. C0-C2 probe site cys330 is highlighted in fuchsia. (C) Ribbon diagram of the THB (PDB: 5K6P) is depicted with locations of the E334K, L349R, and L352P mutations (red). The TMR acceptor labeling site in the THB is indicated (fuchsia triangle).

Table 1.

Effects of C0-C2 mutations on FLT-FRET-based binding to myosin and actin.

C0-C2	PKA	Myosin			Actin
Acceptor Site		C0	C1	THB	C0	C1	THB	Relative to WT FLT-FRET at 2.5 μM
T59A	−	0.91	1.15	0.96	0.97	0.92	1.21#	<0.5
	+	0.80	1.04	1.24	0.89	0.96	0.98	0.8–1.2
R282W	−	1.01	0.98	1.02	1.03	0.97	1.10	1.2–1.5
	+	1.51**	1.24	1.64**	1.37**	1.37**	2.05**	1.5–2.0
E334K	−	1.71**	1.71**	1.96**	1.18*	1.08	1.97**	>2.0
	+	1.67**	1.70**	2.00**	0.45**	1.05	2.03**
L349R	−	1.45**	1.72**	1.60**	1.22**	1.10	1.89**
	+	1.45**	1.43*	1.71**	1.04	1.31**	2.66**
L352P	−	1.46**	1.25#	1.54**	1.36**	1.21**	1.31*
	+	1.36**	0.99	2.01**	2.27**	1.74**	2.60**

Mutations T59A, R282W, E334K, L349R, and L352P in C0-C2 with TMR FRET acceptor in C0 (TMR-C0-C2^cys85), C1 (TMR-C0-C2^cys249), or THB-domain (TMR-C0-C2^cys330) were assessed with 1 μM FMAL-myosin and FMAL-actin. The FRET value of each HCM mutation at 2.5 μM relative to that observed for WT C0-C2 is shown for each probe site. See Fig. S4 for WT FRET values at 2.5 μM C0-C2. The color gradient scheme reflects incremental increases and decreases in FRET values. Red indicates <50% of WT, grey indicates values that are within 20% of WT, light green indicates a 20% to 50% increase, green indicates a 50% to 100% increase, and dark green indicates >100% increase. N ≥ 2 independent biological replicates (protein preparations) and n ≥ 8 technical repeats (wells of binding reactions).

^*p < 0.05

^**p < 0.001, and

^#p ≤ 0.15 for trending towards significant (1-way ANOVA with Dunnett’s post-hoc test) for comparing mutations to WT control.

T59A, in C0, at 2.5 μM TMR-C0-C2, exhibited FRET values that were within 20% of that found for WT TMR-C0-C2 in 4 of the 6 conditions tested (3 probe acceptor sites, minus and plus phosphorylation) for both myosin and actin. For myosin, the probe in THB (cys330) showed a 24% increase in FRET efficiency with myosin when T59A was phosphorylated, however this was not significant (p = 0.5). A 21% increase that was trending in significance (p = 0.09) was seen with actin when unphosphorylated.

R282W, in an M-domain PKA target sequence, displayed little effect on the binding of unphosphorylated R282W to either myosin or actin. Phosphorylated TMR-C0-C2 with R282W displayed significantly increased FRET efficiency with myosin when probes were in C0 and the THB. The 24% increase when the probe was in C1 was not significant (p = 0.3). With actin, 37–105% increases in FRET were significant for all three probe locations. The largest effects on FRET efficiency for phosphorylated R282W with both myosin (64%) and actin (105%) were observed when the acceptor probe was located in the THB at cys330.

E334K, in the THB, increased myosin FRET efficiency in all 6 conditions by 66–100%. With actin, probes on C0 and C1 showed little change (within 18% of WT) and a significant decrease to 45% of WT FRET values for the C0 probe when phosphorylated. When the probe was in the THB, E334K, unphosphorylated and phosphorylated, exhibited twice as much FRET efficiency with actin than WT.

L349R, in the THB, increased myosin FRET values by 43–72% in all conditions tested. Actin FRET efficiency with L349R yielded mixed results with probes on C0 and C1. Minimal change was seen in phosphorylated C0 and unphosphorylated C1 (4% and 10%, respectively). Moderate changes were observed in unphosphorylated C0 and phosphorylated C1 (22% and 31%, respectively). The probe on the THB responded strongly to L349R with increases of 89% (unphosphorylated) and 166% (phosphorylated).

L352P, in the THB, increased FRET efficiency with both myosin and actin. These effects were largest for the probe in THB, followed by the probe in C0 and then in C1. On actin, large effects (74–160%) were observed for all probe sites when L352P was phosphorylated. For myosin, the phosphorylated form showed a large effect (101% increase) on the probe in the THB, a moderate effect (36% increase) on the probe in C0, and no effect on the probe in C1.

4. Discussion

We have developed a relatively simple, high throughput FLT-FRET assay to detect binding of the N-terminal domains of cMyBP-C (C0-C2) to myosin. When used in parallel with a similar assay detecting C0-C2 binding to actin, this makes possible the analysis of multiple C0-C2 conditions and variables including mutations in the presence and absence of PKA phosphorylation on myosin and actin binding.

N-terminal fragments and individual domains of C0-C2 have been used by others to gain insight into their abilities to bind myosin and its fragments. Results from these experiments have not always been consistent. Cardiac and slow skeletal C1-C2 have been reported to bind myosin by using cosedimentation pull-down assays, whereas C0-C1 did not [49]. However, microscale thermophoresis (MST) experiments found that the myosin head (myosin S1 (subfragment 1), lacking RLC binding site, S2 (subfragment 2), and LMM (light meromyosin)) bound equally well to C0-C1 and C0-C2. A “miniHMM” (heavy meromyosin) containing dimeric ΔS2 (the proximal 126 residues of myosin S2) and RLC, but not the myosin S1 head or LMM domains, bound C0-C2 3-fold better than C0-C1, suggesting that binding to ΔS2 and/or RLC is enhanced by the M and C2 domains [50]. Cosedimentation of C1-C2 with myosin was inhibited by ΔS2, suggesting that ΔS2 bound C1-C2 and interfered with its interaction with full-length myosin [49]. Using NMR, isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC), the C0-domain was detected to bind to RLC bound to its myosin binding site (residues 806–963 in myosin heavy chain (MHC)) and also in miniHMM [51]. Phosphorylation, or phosphomimetic mutations, in C1-C2 or C0-C2 were observed to regulate its binding to ΔS2 in yeast two-hybrid, cosedimentation, ITC assays, and in skinned papillary muscle fibers [10,52,53]. Phosphorylation also appeared to regulate the binding of full-length MyBP-C to myosin-S1 heads using MST [39]. In contrast to this result, phosphorylation of C0-C2 did not affect binding to S1 in another MST study [50]. While these techniques (cosedimentation, MST, ITC, NMR, and yeast 2-hybrid) provide valuable insights into cMyBP-C interactions with myosin, they are labor and material intensive, low throughput, and not well-suited for assessing combinations of conditions, variables, and concentrations that can potentially modulate these binding activities.

In the work reported here, we demonstrate that by attaching a donor FRET probe to myosin and an acceptor probe to C0-C2, we can detect C0-C2-myosin binding and differences in that binding due to phosphorylation and mutations in C0-C2. This is done by simply mixing the two labeled proteins in a multi-well (384 wells/plate) format in low volumes (50 μl per sample) at low concentrations (1–2.5 μM) and measuring fluorescence lifetimes in minutes per plate.

4.1. High-throughput myosin-C0-C2 binding FLT-FRET assay

As a proxy for native myofilament interactions, in the myosin-C0-C2 FLT-FRET-based binding assay, we chose to use synthetic thick filaments composed of full-length myosin as this form contains all of the myosin subdomains, such as the RLC, HMM, LMM, S1, and S2 that together with MyBP-C and titin interactions are responsible for maintaining and regulating normal thick filament structure, including stabilization of the interacting-heads motif (IHM) and associated myosin SRX function [54,55]. However, we would expect that other myosin preparations such as soluble HMM or S1 that contain RLC could be readily used as a donor molecule in this assay [17,31,56–59].

C0-C2 constructs with single cysteines (for TMR labeling) located in the C0, C1, or THB of the M-domain ensured that differences in binding due to phosphorylation or cMyBP-C mutations are not due to artifacts of placing the probe in a particular location. It also allowed us to probe potential FRET value differences that result from localizing TMR in a particular domain that might be closer or further away from the FMAL in myosin or actin.

4.2. Myosin versus actin binding to C0-C2

FLT-FRET binding curves for myosin and actin using all three C0-C2 constructs (acceptor probes in C0, C1, and in the THB of the M-domain) showed significant reductions in FRET efficiency upon PKA-mediated phosphorylation. This is consistent with the decreased C0-C2 binding to myosin and to actin observed previously [34,60]. Reductions in FRET efficiency can also be the result of altered modes of binding (e.g., binding that increases the distance or disorder between donor and acceptor probes).

EC₅₀ values of C0-C2 binding to myosin and actin are similar, 1.4–9 μM, though the quantitative order of EC₅₀ values between TMR placed in C0, C1 and the THB are different. In the myosin-C0-C2 FLT-FRET binding assay, the TMR on C1 (C0-C2^cys249) displayed the lowest EC₅₀ (3.4 ± 1.1 μM), followed by TMR on the THB (C0-C2^cys330) (EC₅₀ = 6.9 ± 2.5 μM) and lastly TMR on C0 (C0-C2^cys85) had the highest EC₅₀ (9.0 ± 2.6 μM). The range of EC₅₀ values (3.4–9.0 μM) for myosin binding are consistent with the 2.8–18.2 μM range of K_D values observed by us and others using cosedimentation and MST binding assays [17,46,50]. For actin FLT-FRET assays the order of EC₅₀’s was different, with the lowest EC₅₀ being displayed by TMR on C0 (C0-C2^cys85, EC₅₀ = 1.4 ± 0.2 μM), then C1 (C0-C2^cys249, EC₅₀ = 3.0 ± 0.1 μM), and lastly THB (C0-C2^cys330, 6.3 ± 0.7 μM). The range of EC₅₀ values (1.4–6.3 μM) for actin binding are consistent with the 2.5–13.7 μM range of K_D values observed by us and others using a variety of binding assays [6,34,46]. These results are consistent with the idea that cMyBP-C N-terminal domains bind to both myosin and actin with similar moderate affinity and phosphorylation reduces this affinity and/or alters the arrangement of the cMyBP-C domains relative to myosin and actin filament structures.

4.3. FRET and phosphorylation differences between myosin and actin binding

C0-C2 appears to bind in closer proximity to actin cys374 than to myosin-RLC cys105. FLT-FRET_max values describe the maximum level of FRET efficiency for each binding curve and are dependent on the distance between the FRET donor and acceptor probe and the R₀ of the probe pair. For unphosphorylated C0-C2 levels, myosin FLT-FRET_max values were between ~11–12% FRET efficiency, whereas actin showed values between ~49–56% FRET efficiency. The higher levels of FRET values from C0-C2–actin binding can be interpreted as a closer proximity of the actin filament probe (versus myosin’s RLC probe) to the acceptor probes on C0-C2. It is also possible for C0-C2 bound to neighboring actin monomers to display FLT-FRET with one labeled FMAL-actin and this may further explain the high FRET levels seen for actin binding.

R² values, a measure of the goodness of fit to the quadratic model, are quite good for actin FLT-FRET binding curves for both unphosphorylated and phosphorylated C0-C2 (R² = 0.99–1.00). In the myosin–C0-C2 FLT-FRET binding curves, the unphosphorylated C0-C2 for each probe site indicated that the model was a good fit (R² = 0.93–0.96). This is consistent with previous findings that phosphorylation only somewhat reduces but does not eliminate binding and alters the mode of binding to actin/thin filaments [13,61]. However, the curves generated for phosphorylated C0-C2 in myosin FRET efficiency showed a poor fit with the model (R² = 0.60–0.88). This may be due to the inability to reach FLT-FRET saturation in the phosphorylated C0-C2 binding curves at these concentrations because the affinity is greatly reduced. This is consistent with cosedimentation experiments that found a binding affinity of 2.8 μM for C0-C2 binding to myosin, but reliable fittings were unobtainable for C0-C2 that had phosphomimetic mutations at the PKA phosphorylation sites [17] and with MST binding studies suggesting that phosphorylation of M-domain in N-terminal cMyBP-C fragments nearly eliminates binding to ΔS2 [61].

The relative effects of phosphorylation on cMyBP-C binding to myosin and actin may be critical given the highly localized effective concentration of all 3 proteins arranged in the sarcomere structure [61]. In cardiac muscle, as cMyBP-C is anchored to the thick filament in proximity to interact with both myosin and thin filaments, this greatly increases its effective concentration and likelihood of binding as compared to in vitro studies. cMyBP-C is highly phosphorylated under normal physiological conditions, and it remains unclear how the measured binding properties of freely diffusing N-terminal fragments translate to the binding of full-length cMyBP-C in myocardium. N-terminal cMyBP-C may normally interact with actin and/or myosin despite relatively high phosphorylation levels offset by high effective concentrations in the sarcomere [61], or it may reside mostly in the interfilament space near actin [62]. Phosphorylation levels of cMyBP-C can further increase under β-adrenergic stimulation [63] and this could favor actin over myosin interactions [61]. Consistent with a reserve level of cMyBP-C effects, the population of sequestered myosin and/or myosin with super-relaxed turnover rates are reduced when cMyBP-C normally high phosphorylation levels are further increased [13,17,21,61]. Conversely, reduced phosphorylation in cardiac disease could increase or alter binding compared to normal conditions [23] and this likely affects binding differently for myosin versus actin interactions.

4.4. Probe placement effects on binding curves

The 3–4-fold differences in EC₅₀ values for probes located in different domains may be due to different binding affinities of the different domains of C0-C2 for myosin and actin and/or different modes of binding of the individual domains of C0-C2 containing the FRET acceptor probes. Details of binding are complicated due to multiple binding sites on C0-C2 for myosin or actin [51,64] and multiple binding sites on myosin and actin for C0-C2. Also, bundling of actin filaments indicates that domains within a single C0-C2 molecule can interact independently with multiple actin filaments [6,64]. With maximal actin binding, where there is one C0-C2 bound for each actin monomer, FRET can take place from C0-C2 binding to the labeled actin monomer or from C0-C2 FRET on neighboring actin monomers. Thus, the different EC₅₀s may be due to FRET efficiency being a measure of both overall binding and where individual binding site events take place.

For actin, we have examined overall binding by cosedimentation assays where the combined interactions of all C0-C2 domains are detected as actin binding. Examination of the binding of TMR-C0-C2 with FMAL-actin by cosedimentation suggests that both altered overall affinities and altered modes of binding of individual domains are factors in the FLT-FRET assay. Cosedimentation using unphosphorylated TMR-C0-C2 and FMAL-actin displayed differences in EC₅₀ values that were similar to that observed in the FLT-FRET assay, with EC₅₀ values C0-C2^cys85 < C0-C2^cys249 < C0-C2^cys330. There was good correlation between the two assays (Fig. S3). This suggests that the FLT-FRET differences in binding EC₅₀s are due to overall binding changes resulting from placing probes in different domains. In cosedimentation assays, phosphorylation of C0-C2^cys85, C0-C2^cys249, and C0-C2^cys330, resulted in a small decrease in overall binding. EC₅₀s were increased 1.1 × (C0-C2^cys85), 1.6 × (C0-C2^cys249), and 1.1 × (C0-C2^cys330). The 1.6 × change in overall binding by C0-C2^cys249 was mirrored by a similar change of 1.7 × in the FLT-FRET assay. For probes in C0 (C0-C2^cys85) and the THB (C0-C2^cys330), even though very small changes in cosedimentation reported overall binding (1.1 ×) were observed, the FRET efficiency changes due to phosphorylation were large (2.9 × and 2.8 × changes in EC₅₀). This suggests that although FRET-based binding of unphosphorylated C0-C2 to actin was similar overall (to cosedimentation data), the positions of C0 and the THB relative to actin changed such that they were further away from the FMAL on actin when TMR-C0-C2 is phosphorylated.

Due to problems with traditional binding assays that depend on assessing FLT-FRET_max (or B_max for cosedimentation), we have argued that a better comparison is to observe values at submaximal levels [34]. For the analysis of mutant effects, we focused on 2.5 μM TMR-C0-C2. The FRET effects upon binding to actin are similar for 2.5 μM C0-C2^cys85 and C0-C2^cys249 and reduced by 52% upon phosphorylation. TMR-C0-C2^cys330 FRET efficiency was about half that observed for the other 2 probe sites and the effect of phosphorylation was a 67% reduction in FRET efficiency. The effects of the M-domain on muscle function are mediated by both the phosphorylated region and the THB [21] and the results of using either the values at 2.5 μM or EC₅₀s for comparison of FRET effects due to phosphorylation, when the probe is in the THB (TMR-C0-C2^cys330), illustrates the importance of the THB. By comparison, cosedimentation at 2.5 μM C0-C2 indicate similar overall binding between the different C0-C2 proteins when not phosphorylated and smaller decreases (26–40%) upon phosphorylation. The large TMR-C0-C2^cys330 FRET changes (67% upon phosphorylation) are reduced to 33% in cosedimentation values. As was seen when using EC₅₀ values for comparison, cosedimentation demonstrates that the interactions of the THB with actin is more sensitive to phosphorylation than overall binding of C0-C2. The EC₅₀ comparisons, but not those at 2.5 μM, suggest that the same is true for the binding of C0 that is separated from the phosphorylated M-domain by the PAL and C1. Altered positioning of C0 is consistent with experiments that found changes in C0-C1 spatial relationships upon phosphorylation of C0-C2 [18]. Therefore, the FLT-FRET assay displayed sensitivity to the mode of binding that is missing in cosedimentation assays. For actin, differential effects of unphosphorylated MyBP-C versus phosphorylated binding have been noted previously [13,61].

Myosin cosedimentation experiments are beyond the scope of this work but from the work with actin we speculate that differences in the myosin FLT-FRET binding curves with unphosphorylated C0-C2 may be due to the presence of TMR in different domains of C0-C2. Phosphorylated C0-C2 binding to myosin did not fit well to the quadratic model as indicated by poor adj. R² values (0.60–0.88) (Table S1), consistent with the idea that interactions between phosphorylated C0-C2 and myosin are greatly reduced, if not completely abolished.

4.5. Effects of 5 C0-C2 mutations on myosin and actin binding

Five HCM-linked or function-altering mutations were tested in each of the 3 TMR-C0-C2 FRET acceptor proteins to determine whether they affect binding to myosin, actin, or both, and whether these changes persist with or without phosphorylation. These comparisons can suggest specific mechanisms of pathological dysfunction. The FLT-FRET results at 2.5 μM of C0-C2 binding to myosin and actin is below maximal binding in order to readily detect potential increases or decreases in FRET efficiency for all 3 probes located in the C0, C1, and THB of the M-domain (arrowheads in Fig. 3 and Fig. S5–S8), indicating changes in binding and/or binding mode. The mutations tested were in the C0-domain (T59A) and the M-domain (R282W, in a PKA phosphorylation recognition site, and E334K, L349R, L352P in the THB). PKA phosphorylation of all HCM mutants, except for R282W, showed normal phosphorylation levels compared to WT (Fig. S9). We previously determined that R282W has reduced phosphorylation levels due to the mutation residing in the PKA recognition site of S284 [34]. These mutations were chosen since they are in the phosphorylatable M-domain and have been previously shown to alter binding of cMyBP-C to actin and/or myosin, with the exception of T59A. Mechanistically, it is unknown how these mutations affect binding to myosin and actin and how cMyBP-C phosphorylation affects this binding.

T59A was identified in a survey of sarcomeric protein gene mutations in HCM [40] and is listed in ClinVar as a pathogenic variant. We chose to study this mutation partially due to its reported pathogenicity. However, this variant has only been reported once to ClinVar, and may not be conclusively linked to familial HCM [40]. Although T59A replaces the hydrophilic polar threonine with the hydrophobic nonpolar alanine, it has been suggested to be benign as alanine is found in this position in other species [51]. T59A showed the smallest effects in both myosin and actin FLT-FRET binding assays of the 5 mutations tested. For all 3 probe locations, T59A showed less that a 25% change from WT in either the unphosphorylated or phosphorylated state (Table 1). These results are consistent with an interpretation in that T59A is benign for C0-C2 interactions with myosin or actin.

R282W was identified in a survey of cMyBP-C mutations associated with HCM [41]. This variant changes one of the PKA target recognition sequences (281–284) from RRIS to RWIS (Fig. 4), eliminating PKA phosphorylation of S284, and leads to altered structural dynamics [19,21]. Previous actin cosedimentation assays showed that unphosphorylated R282W had no change in binding to actin or actin-tropomyosin while phosphorylated R282W, lacking phosphorylation at S284, showed increased actin binding, indicating that phosphorylation of the remaining 3 PKA targets (S275, S304, and S311) was not sufficient to elicit the maximal PKA effect [34]. PKA phosphorylation of R282W in C0-C2 was significantly reduced compared to WT (Fig. S9), as we observed previously [34]. This serine has also been demonstrated to be key to the PKA-mediated reduction in C0-C2 binding to myosin [17]. The results of our FLT-FRET assays agree with these findings. No change in the binding of unphosphorylated R282W and increased interactions of PKA-treated R282W compared to WT for both myosin and actin was observed. The largest increase in FRET efficiency was observed for the probe in the THB suggesting that reduction in phosphorylation in the disordered region of the M-domain has a strong influence on the associations of the THB with actin (Table 1). Although the in vitro techniques used here cannot determine pathogenicity, our previous data [34] and the data obtained from these new FRET assays suggest that if R282W is pathogenic, this is likely due to its reduced capacity to be phosphorylated. This reduced effect of phosphorylation alters interactions with both myosin and actin.

THB mutations: E334K, L349R, and L352P mutations are in the THB region, in the C-terminal region of the M-domain (Fig. 4), and these mutations generally displayed increased binding to myosin and actin in our FLT-FRET binding assays. The third helix of the THB has been identified as critical to actin binding. Cryo-EM analysis indicate that the positively charged residues on the surface of helix 3 (R346, K350 and K353) interact with actin [21,64]. L349 and L352 are in helix 3 but are not predicted to interact with myosin or actin as they are buried in the hydrophobic core of the THB. Their mutation likely destabilizes the THB allowing for altered binding of helix 3 to actin and possibly myosin [11]. E334, in helix 2, also appears to not be involved in interactions with actin and has been postulated to stabilize helix 2 and the THB fold. E334K is also predicted to destabilize the THB [21]. It has been noted previously that increased binding brought about by opening of the THB might occur under normal physiological conditions if the M-domain is stretched during contraction [65]. The THB may itself be regulated by phosphorylation of the neighboring disordered region in the M-domain, as suggested by molecular dynamics simulations [18].

E334K has been associated with HCM [42,66] and is predicted to impact secondary protein structure. Reduced actin binding was observed in mouse E334K C1-C2 [21]. E334K in human C0-C2 did not display changes in actin binding in FLT-based assays and slightly increased binding in cosedimentation assays [34]. Mutation of the homologous residue in slow skeletal MyBP-C increased interactions with myosin [67]. Consistent with these results, all 3 FRET probes in cMyBP-C indicated binding of unphosphorylated and phosphorylated TMR-C0-C2 to myosin was increased. Interactions detected between actin and the probe in C1 for E334K resulted in unchanged FRET efficiency in both unphosphorylated and phosphorylated TMR-C0-C2, while FRET efficiency from the probe in C0 was not affected in unphosphorylated TMR-C0-C2 but was reduced by half in the phosphorylated state. This suggests that, just as C0 alters the positioning of C1 on actin [12], the THB, where E334K is located, can influence the positioning of C0. These results suggest a repositioning of the N-terminal domains of cMyBP-C, which may enhance myosin but not actin binding. However, this is complicated by interactions observed from the probe located in the THB that resulted in FRET values twice that seen for WT TMR-C0-C2, similar to the increase observed for myosin. These results with actin, slight increases in overall binding observed in cosedimentation assays and 2-fold increases and decreases with probes in different domains, indicate that E334K may also affect actin interactions. That a larger FRET effect is observed for this mutation when the probe is in the THB is perhaps not surprising as the mutation is just 4 amino acids from the probe in the THB that appears to bind actin [21].

L349R (L263R in slow skeletal MyBP-C) has been reported to decrease binding of M-domain to myosin in the slow skeletal MyBP-C isoform resulting in skeletal muscle weakness and tremor [43]. We observed increases of 45–72% in binding of L349R in the myosin-C0-C2 FLT-FRET binding assay for all 3 probe positions with or without PKA phosphorylation. Differences between our results and those observed for slow skeletal binding to HMM may be due to isoform or technical differences. Actin binding was either not affected or increased by 20–30% as reported by probes in C0 and C1. The probe in the THB displayed very significant increases in FRET efficiency with actin, ~89% when unphosphorylated and ~166% when phosphorylated. The ~2-fold difference in the magnitude of effect (between unphosphorylated and phosphorylated L349R) for the probe in the THB, again, indicates the likely importance of phosphorylation on the interactions between the THB and actin. As with E334K, the larger increases in FRET efficiency for C0 and C1 probe placements binding to myosin over actin may indicate enhanced myosin binding with this mutation.

L352P was identified as an HCM mutation [68]. Using FLT and cosedimentation binding assays, we and others have reported increased actin/thin filament binding of cMyBP-C containing the L352P mutation with greater effects seen in phosphorylated than the unphosphorylated C0-C2 [34,44]. Consistent with these results, actin binding of L352P with probes in C0, C1 and THB all displayed 21–36% increased FRET efficiency when unphosphorylated and 74–160% increased FRET values when phosphorylated, with the site in the THB, again, showing the greatest increase. For myosin binding, all three probe locations displayed increases in FRET efficiency (25–100%) without a clear pattern for phosphorylated versus unphosphorylated. These results suggest that actin binding is enhanced by L352P, as expected [34,44], but interestingly, this effect of enhanced binding is even greater in the phosphorylated state.

Enhanced myosin and actin binding of E334K, L349R, and L352P HCM variants in cMyBP-C may contribute to the disease pathology by altering thick and/or thin filament structural arrangements and cross-bridge cycling properties. A hallmark characteristic of HCM is hypercontractility of the heart, often linked to HCM-causing sarcomeric mutations. Hypercontractility may arise from various determinants of muscle force (and work) and enhanced interactions of myosin and actin with HCM-mutant cMyBP-C could affect inherent functions of the myofilament. cMyBP-C has been shown to affect the number of cycling myosin heads [59,69], the rate of cross-bridge cycling [70], and sensitivity of the thin filaments for myosin binding [71]. cMyBP-C mutations could also conceivably affect the myosin duty ratio, force per cross-bridge and/or step size, although these effects have not yet been directly demonstrated to our knowledge. Any deviation from normal myofilament function in HCM variants, including those leading to enhanced (or reduced) myosin- or actin-MyBP-C binding in vitro, could have dysregulated and deleterious effects in vivo that manifest directly or due to compensatory mechanisms to facilitate hypercontractility. Changes deviating from normal myosin/actin interactions with cMyBP-C could lead to dysfunction on the whole heart level over the course of the disease progression. Additional studies, including mechanical measurement of force in myocardium containing these HCM variants of MyBP-C can provide key insights.

This group of mutants, largely localized to the M-domain, generally displayed increased interactions with myosin and actin in the unphosphorylated and especially in the phosphorylated states. Increased interactions are also predicted to result from decreased phosphorylation levels (in the absence of HCM mutations in cMyBP-C) that are observed during the development of heart failure, pathologic hypertrophy, and ischemia-reperfusion injury and HCM caused by mutations in other sarcomere proteins [23,72]. In both the mutants studied here and in hypophosphorylation, increased in vitro interactions are associated with pathological states.

The two distinct regions comprising the M-domain, a disordered region where phosphorylation occurs and an ordered THB region, both maintain interactions with actin [64]. Our results demonstrate the importance of each of these regions with R282W influencing the disordered region in the phosphorylated state and E334K, L349R, and L352P impacting the THB. Our results also demonstrate the additive effects of the THB and the disordered phosphorylated regions as mutations in the THB are observed in both the phosphorylated and unphosphorylated states with neither mutations nor phosphorylation negating the other’s effects. Similar dual interactions of M-domain may be occurring with myosin given the parallel additive effects of mutations and phosphorylation on FLT-FRET reported binding.

4.6. Potential of FLT-FRET myosin-C0-C2 and actin-C0-C2 binding assays in HTS

Having validated both the myosin and actin binding FLT-FRET assays’ abilities to detect binding, we quantitated their usefulness in HTS. $Z^{\prime }$ is a measure of the suitability of conditions for conducting HTS for compounds that precisely change signal as an indication of change from one state to another. When the difference in signal between the two states is greater than the sum of 3 standard deviations from each state, then the $Z^{\prime }$ is >0 and suggests that conditions are appropriate for HTS. When the difference in signal between the 2 states is twice the sum of 3 standard deviations from each state, then the $Z^{\prime }$ is >0.5 and conditions are excellent for HTS [35,48] (Eq. 3, Material and Methods). In the present study, Table S3 shows the $Z^{\prime }$ values of 1 μM myosin $\left(Z^{\prime }=0.76-0.90\right)$ and 1 μM actin $\left(Z^{\prime }=0.94-0.95\right)$ in the absence and presence of 5 μM C0-C2^cys249 (black arrows in Fig. 3), indicating excellent conditions for HTS of compounds that alter C0-C2 binding to myosin or actin. Similarly robust $Z^{\prime }$ were obtained for all probe locations and also when comparing phosphorylated and unphosphorylated TMR-C0-C2 (not shown, but clear from the curves in Fig. 3). While this work was being conducted, we used FMAL-actin and TMR-C0-C2^cys249 in a screen to identify 32 compounds (from a 2684 compound library) that either decreased or increased actin-C0-C2 interactions [36]. Based on the assay $Z^{\prime }$ (Table S3), we expect to be able to screen for compounds affecting myosin-C0-C2 interactions.

4.7. Limitations of the current study

The specific details and relevance of our observed binding properties based on FLT-FRET and cosedimentation assays reported here need to be viewed with caution as they represent interactions of N-terminal domains in solution with myosin or actin, which may not fully depict binding properties in the intact sarcomeres of myocardium. Investigations of full-length cMyBP-C variants incorporated into myocardium would provide complementary details of how altered binding leads to HCM dysfunction. Additionally, we have examined conditions where C0-C2 is unphosphorylated and maximally phosphorylated while in normal human tissue is approximately 65% phosphorylated and this varies with condition and pathology. Additional work, as we have done for R282W [34], is need determine the effects of mutations on intermediate levels of phosphorylation. Finally, the general observation of increased binding activity due to the mutations we have examined in the M-domain may or may not be the case for other HCM mutations in other domains.

4.8. Conclusions and future directions

Here, we developed complementary, high-throughput, cMyBP-C N-terminal FLT-FRET-based binding assays for myosin and actin. These assays were then used to characterize the effects on myosin and actin binding of 5 HCM-linked or functional mutations (T59A, R282W, E334K, L349R, and L352P) in unphosphorylated and phosphorylated C0-C2. The assays showed (1) phosphorylation decreases binding to both myosin and actin, (2) HCM mutations in the M-domain largely increase binding to both myosin and actin, (3) the mutation effects are greatest in phosphorylated C0-C2, and (4) some mutations have a larger impact on actin than myosin binding. All of these new findings support a mechanism where MyBP-C can normally interact with myosin and actin and that phosphorylation and HCM mutations can perturb binding. (5) Phosphorylation of M-domain resulted in modest changes in actin binding in cosedimentation assays that are based on overall C0-C2-actin binding, and larger changes detected in the FLT-FRET assays, indicate that these measurements are based on overall binding plus the spatial relationship of the probes on C0-C2 and actin. (6) Thus, the magnitude of the phosphorylation dependent changes measured by FLT-FRET were dependent on C0-C2 probe location (C0, C1, or M-domain). These findings support a model where, in addition to changes in N-terminal binding between myosin and actin, phosphorylation and mutations change the mode of binding, such as which of the multiple binding interactions mediated by different cMyBP-C domains are taking place. This is supported by our earlier studies where we detected very different actin rotational dynamics between bound phosphorylated and unphosphorylated cMyBP-C N-terminal domains [13]. Further, the opposite effects of phosphorylation and M-domain mutations in cMyBP-C is consistent with the idea that phosphorylation to reduce binding is normally high and cardioprotective, as 3 of the 4 M-domain mutants that we tested are associated with HCM and increased binding. Finally, we quantitated the precision of these assays for their usefulness in future high-throughput screening of drug libraries for targeting MyBP-C as new therapy. These complementary high-throughput FLT-FRET assays enhance our capacity to test combinatory variables that impact the concentration-dependent phosphorylation-regulated myosin and actin binding of C0-C2 in healthy and mutant diseased states and set the stage to screen for drugs to modulate those interactions as new therapeutic interventions.

Abbreviations:

cMyBP-C

Cardiac myosin-binding protein C

FMAL

fluorescein maleimide

FLT

fluorescence lifetime

FRET

fluorescence resonance energy transfer

HF

heart failure; HTS, high-throughput screening

HCM

hypertrophic cardiomyopathy

M-domain

motif domain

RLC

myosin regulatory light chain

SRX

myosin super-relaxed state

C0-C2

N-terminal domains C0 through C2

PKA

protein kinase A

TMR

tetramethylrhodamine

THB

tri-helix bundle