Title: The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner
Abstract: Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (Kd) of ∼10 μm and a molar binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced Bmax) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling. Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (Kd) of ∼10 μm and a molar binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced Bmax) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling. Cardiac myosin-binding protein C (cMyBP-C) 2The abbreviations used are: cMyBP-C, cardiac myosin-binding protein C; NTF, native thin filaments; PKA, protein kinase A; DTT, dithiothreitol. is a thick filament accessory protein that performs both structural and regulatory functions within vertebrate sarcomeres. Both roles are likely to be essential in deciphering how a growing number of mutations found in the cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure in a substantial number of the world's population (1Richard P. Charron P. Carrier L. Ledeuil C. Cheav T. Pichereau C. Benaiche A. Isnard R. Dubourg O. Burban M. Gueffet J.P. Millaire A. Desnos M. Schwartz K. Hainque B. Komajda M. Circulation. 2003; 107: 2227-2232Crossref PubMed Scopus (1010) Google Scholar, 2Dhandapany P.S. Sadayappan S. Xue Y. Powell G.T. Rani D.S. Nallari P. Rai T.S. Khullar M. Soares P. Bahl A. Tharkan J.M. Vaideeswar P. Rathinavel A. Narasimhan C. Ayapati D.R. Ayub Q. Mehdi S.Q. Oppenheimer S. Richards M.B. Price A.L. Patterson N. Reich D. Singh L. Tyler-Smith C. Thangaraj K. Nat. Genet. 2009; 41: 187-191Crossref PubMed Scopus (218) Google Scholar). Considerable progress has recently been made in determining the regulatory functions of cMyBP-C and it is now apparent that cMyBP-C normally limits cross-bridge cycling kinetics and is critical for cardiac function (3Harris S.P. Bartley C.R. Hacker T.A. McDonald K.S. Douglas P.S. Greaser M.L. Powers P.A. Moss R.L. Circ. Res. 2002; 90: 594-601Crossref PubMed Scopus (281) Google Scholar, 4Korte F.S. McDonald K.S. Harris S.P. Moss R.L. Circ. Res. 2003; 93: 752-758Crossref PubMed Scopus (135) Google Scholar, 5Stelzer J.E. Fitzsimons D.P. Moss R.L. Biophys. J. 2006; 90: 4119-4127Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Phosphorylation of cMyBP-C is essential for its regulatory effects because elimination of phosphorylation sites (serine to alanine substitutions) abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge cycling kinetics and blunts cardiac responses to inotropic stimuli (6Tong C.W. Stelzer J.E. Greaser M.L. Powers P.A. Moss R.L. Circ. Res. 2008; 103: 974-982Crossref PubMed Scopus (156) Google Scholar). The substitutions further impair cardiac function, reduce contractile reserve, and cause cardiac hypertrophy in transgenic mice (6Tong C.W. Stelzer J.E. Greaser M.L. Powers P.A. Moss R.L. Circ. Res. 2008; 103: 974-982Crossref PubMed Scopus (156) Google Scholar, 7Nagayama T. Takimoto E. Sadayappan S. Mudd J.O. Seidman J.G. Robbins J. Kass D.A. Circulation. 2007; 116: 2399-2408Crossref PubMed Scopus (67) Google Scholar). By contrast, substitution of aspartic acids at these sites to mimic constitutive phosphorylation is benign or cardioprotective (8Sadayappan S. Osinska H. Klevitsky R. Lorenz J.N. Sargent M. Molkentin J.D. Seidman C.E. Seidman J.G. Robbins J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16918-16923Crossref PubMed Scopus (168) Google Scholar). Although a role for cMyBP-C in modulating cross-bridge kinetics is supported by several transgenic and knock-out mouse models (6Tong C.W. Stelzer J.E. Greaser M.L. Powers P.A. Moss R.L. Circ. Res. 2008; 103: 974-982Crossref PubMed Scopus (156) Google Scholar, 7Nagayama T. Takimoto E. Sadayappan S. Mudd J.O. Seidman J.G. Robbins J. Kass D.A. Circulation. 2007; 116: 2399-2408Crossref PubMed Scopus (67) Google Scholar, 9Palmer B.M. Georgakopoulos D. Janssen P.M. Wang Y. Alpert N.R. Belardi D.F. Harris S.P. Moss R.L. Burgon P.G. Seidman C.E. Seidman J.G. Maughan D.W. Kass D.A. Circ. Res. 2004; 94: 1249-1255Crossref PubMed Scopus (95) Google Scholar, 10Stelzer J.E. Patel J.R. Walker J.W. Moss R.L. Circ. Res. 2007; 101: 503-511Crossref PubMed Scopus (137) Google Scholar), the precise mechanisms by which cMyBP-C exerts these effects are not completely understood. For instance, the unique regulatory motif or “m-domain” of cMyBP-C binds to the S2 subfragment of myosin in vitro (11Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (200) Google Scholar) and binding is abolished by PKA-mediated phosphorylation of the m-domain (12Gruen M. Prinz H. Gautel M. FEBS Lett. 1999; 453: 254-259Crossref PubMed Scopus (159) Google Scholar). These observations have led to the idea that (un)binding of the m-domain from myosin S2 mediates PKA-induced increases in cross-bridge cycling kinetics. Consistent with this idea, Calaghan and colleagues (13Calaghan S.C. Trinick J. Knight P.J. White E. J. Physiol. 2000; 528: 151-156Crossref PubMed Scopus (40) Google Scholar) showed that S2 added to transiently permeabilized myocytes increased their contractility, presumably because added S2 displaced cMyBP-C from binding endogenous S2. However, other reports indicate that cMyBP-C can influence actomyosin interactions through mechanisms unrelated to S2 binding, because either purified cMyBP-C (14Saber W. Begin K.J. Warshaw D.M. VanBuren P. J. Mol. Cell Cardiol. 2008; 44: 1053-1061Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) or recombinant N-terminal domains of cMyBP-C (15Shaffer J.F. Razumova M.V. Tu A.Y. Regnier M. Harris S.P. FEBS Lett. 2007; 581: 1501-1504Crossref PubMed Scopus (25) Google Scholar) affected acto-S1 filament sliding velocities and ATPase rates in the absence of myosin S2. These results thus raise the possibility that interactions with ligands other than myosin S2, such as actin or myosin S1, contribute to effects of cMyBP-C on cross-bridge interaction kinetics. The idea that cMyBP-C interacts with actin to influence cross-bridge cycling kinetics is supported by several studies that implicate the regulatory m-domain or sequences near it in actin binding (16Whitten A.E. Jeffries C.M. Harris S.P. Trewhella J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 18360-18365Crossref PubMed Scopus (101) Google Scholar, 17Squire J.M. Luther P.K. Knupp C. J. Mol. Biol. 2003; 331: 713-724Crossref PubMed Scopus (135) Google Scholar, 18Razumova M.V. Shaffer J.F. Tu A.Y. Flint G.V. Regnier M. Harris S.P. J. Biol. Chem. 2006; 281: 35846-35854Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Kulikovskaya I. McClellan G. Flavigny J. Carrier L. Winegrad S. J. Gen. Physiol. 2003; 122: 761-774Crossref PubMed Scopus (109) Google Scholar). cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 11 repeating domains that bear homology to either Ig or fibronectin-like folds. Domains are numbered sequentially from the N terminus of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100 amino acids, is located between domains C1 and C2 and is phosphorylated on at least 3 serine residues by PKA (12Gruen M. Prinz H. Gautel M. FEBS Lett. 1999; 453: 254-259Crossref PubMed Scopus (159) Google Scholar). Although the precise structure of the m-domain is not known, small angle x-ray scattering data suggest that it is compact and folded in solution and is thus similar in size and dimensions to the surrounding Ig domains (20Jeffries C.M. Whitten A.E. Harris S.P. Trewhella J. J. Mol. Biol. 2008; 377: 1186-1199Crossref PubMed Scopus (58) Google Scholar). Recombinant proteins encompassing the m-domain and/or a combination of adjacent domains including C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been shown to bind actin (16Whitten A.E. Jeffries C.M. Harris S.P. Trewhella J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 18360-18365Crossref PubMed Scopus (101) Google Scholar, 18Razumova M.V. Shaffer J.F. Tu A.Y. Flint G.V. Regnier M. Harris S.P. J. Biol. Chem. 2006; 281: 35846-35854Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Kulikovskaya I. McClellan G. Flavigny J. Carrier L. Winegrad S. J. Gen. Physiol. 2003; 122: 761-774Crossref PubMed Scopus (109) Google Scholar). The purpose of the present study was to characterize binding interactions of the N terminus of cMyBP-C with actin and to determine whether interactions with actin are influenced by phosphorylation of the m-domain. Results demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin filaments with affinities similar to that reported for cMyBP-C binding to myosin S2 (11Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (200) Google Scholar). Furthermore, actin binding was reduced by m-domain phosphorylation, suggesting that reversible interactions of cMyBP-C with actin could contribute to modulation of cross-bridge kinetics. Protein Expression and Purification—Recombinant murine cMyBP-C proteins containing various combinations of N-terminal domains with and without the m-domain were cloned and purified as described previously (18Razumova M.V. Shaffer J.F. Tu A.Y. Flint G.V. Regnier M. Harris S.P. J. Biol. Chem. 2006; 281: 35846-35854Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 21Razumova M.V. Bezold K.L. Tu A.Y. Regnier M. Harris S.P. J. Gen. Physiol. 2008; 132: 575-585Crossref PubMed Scopus (40) Google Scholar). In some cases proteins were further purified by ion exchange chromatography (Bio-Rad). All proteins were centrifuged for 20 min at 120,000 rpm (511,000 × g) and 4 °C in a TLA 120.2 rotor in an Optima TLX ultracentrifuge (Beckman Coulter, Fullerton, CA) prior to use to remove any insoluble material. Final protein concentration was determined by UV spectrometry using extinction coefficients calculated from amino acid primary sequences and software from the Swiss Institute of Bioinformatics website (22Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic Acids Res. 2003; 31: 3784-3788Crossref PubMed Scopus (3480) Google Scholar). At least two separate preparations of recombinant cMyBP-C proteins were used for each experimental data set. Rabbit skeletal myosin S1 was prepared by chymotryptic digestion of myosin in the presence of EDTA as described (23Margossian S.S. Lowey S. Methods Enzymol. 1982; 85: 55-71Crossref PubMed Scopus (825) Google Scholar). Myosin S2Δ (comprised of the N-terminal 126 amino acids of myosin S2 (11Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (200) Google Scholar)) was prepared by reverse transcriptase-PCR of RNA isolated from rat heart and subsequent cDNA cloning into the pQE-2 expression vector (Qiagen, Valencia, CA). Because the low percentage of aromatic amino acids in the S2Δ sequence precludes accurate measurement of concentration by UV spectrometry, the concentration of purified S2Δ was determined by amino acid analysis performed by the University of California Davis Molecular Structure Facility. Bovine cardiac F-actin was prepared from ether powder as described (24Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (982) Google Scholar). F-actin was maintained in a storage buffer (in mmol/liter: 50 KCl, 1 MgCl2, 2 Tris-HCl, pH 8.0, 0.2 CaCl2, 0.5 β-mercaptoethanol, 1 ATP, and 0.02% sodium azide) and kept at 4 °C until use. Native thin filaments (NTF) were purified from bovine heart left ventricle according to a modification of the protocol by Spiess et al. (25Spiess M. Steinmetz M.O. Mandinova A. Wolpensinger B. Aebi U. Atar D. J. Struct. Biol. 1999; 126: 98-104Crossref PubMed Scopus (14) Google Scholar). Four volumes of buffer (in mmol/liter: 10 KPO4, pH 7.0, 100 KCl, 5 MgCl2, 1 EGTA, 1 NaN3, 1 DTT, and 1% Triton X-100) were added to ventricle minced in a Waring grinder and the tissue was homogenized using a Polytron PT3100. Protease inhibitor mixture (in mg/ml: 0.01 phenylmethylsulfonyl fluoride, 0.001 leupeptin, 0.001 pepstatin, 0.001 antipain) was added and the solution spun at 3,800 rpm for 13 min with a JS-4.2 rotor (Beckman). The pellet was homogenized, resuspended, and centrifuged again 4 more times with the last 3 in buffer lacking Triton X-100. Next, the pellet containing myofibrils was homogenized in 1.6 volumes of buffer plus 5 mm ATP to dissociate the thin and thick filaments. The solution was spun as above and the pellets (containing mostly thick filaments) discarded. The supernatant was then spun at 70,000 rpm for 15 min (Beckman Type 70 Ti rotor), the pellet discarded, then the supernatant spun again for 80 min. The pellets (containing mostly thin filaments) were resuspended in 25 ml of dialysis buffer (in mmol/liter: 20 Tris-HCl, 100 KCl, 5 MgCl2, 1 EGTA, 1 NaN3, 1 DTT) using a Dounce homogenizer and dialyzed overnight. The following day, the solution was brought to 200 mm KCl and 5 mm ATP and spun at 45,000 rpm for 15 min (Beckman Type 50.2 rotor) to remove any contaminating myosin. Pellets were discarded, and the supernatant was spun again for 150 min. The pellets were resuspended as above and dialyzed overnight. The purification cycle (centrifugation for 15 min, pellet discard, and centrifugation for 150 min) was repeated again the next day. Final pellets were overlaid with NTF buffer (in mmol/liter: 5 imidazole, pH 7.0, 50 KCl, 2 MgCl2, 0.2 ATP, 0.5 DTT) overnight to soften. The pellets were resuspended gently with a pipette and spun at 19,500 rpm for 30 min (Beckman JA-20 rotor) to remove debris and aggregated material. NTF concentration was measured by UV spectrometry using an extinction coefficient (280 nm) = 0.0529 μm-1 cm-1 and molecular weight = 62,500 per actin for the thin filament complex (7:1:1, actin:tropomyosin:troponin). Ca2+ regulation of purified NTF was confirmed prior to use in cosedimentation assays by measuring actin-activated S1-ATPase rates as described previously (15Shaffer J.F. Razumova M.V. Tu A.Y. Regnier M. Harris S.P. FEBS Lett. 2007; 581: 1501-1504Crossref PubMed Scopus (25) Google Scholar) except buffers contained 1 mm EGTA. Activity was 0.2 ± 0.1 s-1 in the absence of Ca2+ and 3.3 ± 0.9 s-1 (n = 6) in the presence of Ca2+ (pCa 3). Phosphorylation of recombinant C1C2 was achieved by incubation with the catalytic subunit of PKA (Sigma P2645). C1C2 was dialyzed against a buffer containing (in mmol/liter: 20 HEPES, pH 7.4, 100 KCl, 10 MgCl2, 1 ATP, and 1 DTT). PKA was resuspended in 6 mg/ml DTT per the manufacturer's instructions and combined with an additional 1 mm ATP and C1C2 at 40 units of PKA/mg of C1C2. The reaction mixture was incubated at 4 °C for >5 h and then applied to a nickel-nitrilotriacetic acid column (Qiagen) to purify phosphorylated C1C2 and remove the PKA catalytic subunit. Phosphorylation status was assessed by Pro Q Diamond staining followed by Sypro Ruby staining (Invitrogen). Cosedimentation Assays—Recombinant cMyBP-C proteins were dialyzed against a cosedimentation buffer (in mmol/liter: 20 imidazole, pH 7.4, 180 KCl, 1 MgCl2, 1 EGTA, 1 DTT). F-actin remained in its storage buffer prior to use. Recombinant cMyBP-C proteins (1-30 μm final concentrations) were combined with sufficient F-actin, ATP, and DTT to achieve final concentrations of 5 μm, 1 mm, and 1 mm, respectively, in a total final volume of 50 μl of cosedimentation buffer. For experiments with S2Δ, a 6-fold excess of S2Δ (30 μm) to actin was added to each tube. Reactions were allowed to equilibrate for 30 min at room temperature. Samples were then spun for 30 min at 100,000 rpm (390,000 × g) using a TLA 100 rotor at 4 °C (Beckman). The supernatants were removed from each tube and the pellets gently washed with 50 μl of cosedimentation buffer and dissolved in 100 μl of a 1:1 mixture of cosedimentation buffer to urea/thiourea sample buffer (26Fritz J.D. Swartz D.R. Greaser M.L. Anal. Biochem. 1989; 180: 205-210Crossref PubMed Scopus (232) Google Scholar). Pellet fractions were loaded onto 10% polyacrylamide gels and run at constant 200 V for 50 min. Gels were stained for at least 1 h in 0.05% Coomassie R-250 staining solution, followed by destaining and drying overnight. Quantification of Binding Data—Dried gels were scanned to a computer using a flatbed scanner and band intensities were measured using the gel analysis features of Image J (NIH, Bethesda, MD). The intensity ratio of recombinant protein to F-actin in each pellet was converted to a molar ratio (mole of cMyBP-C/mole of actin) using standard curves run on each gel that contained known amounts of cMyBP-C and actin in mol/mol ratios (Fig. 1). Standard curves were constructed by adding 40 pmol of actin to each of 7 tubes followed by addition of increasing amounts of recombinant protein (4-40 pmol) to achieve the desired mole ratio. A fixed volume from each standard tube (3.5 μl) was loaded onto the gel so that the amount of actin typically varied from 13 to 7.8 pmol across the wells of the standard curve. This range was selected to verify that staining intensity was linear over the amount of actin (10.6 pmol) loaded in each cosedimentation assay. Cosedimentation binding data were plotted versus the total cMyBP-C concentration added and fit according to the following equation (27Wilkinson K.D. Methods Mol. Biol. 2004; 261: 15-32PubMed Google Scholar), [Cbound][Actin] = Bmax[Cfree]Kd + [Cfree](Eq. 1) where [Cbound] and [Cfree] are the bound and free concentrations of cMyBP-C proteins, respectively, [Actin] is the total actin concentration, Bmax is the maximal molar binding ratio (mole of cMyBP-C protein/mole of actin), and Kd is the dissociation constant (μm). Equation 1 can be rearranged by substituting Equation 2 in for [Cfree] and solving for [Cbound]/[Actin] to fit the data as a function of total cMyBP-C protein added ([Ctotal]), as shown in Equation 3. [Ctotal] = [Cbound] + [Cfree](Eq. 2) [Cbound ][ Actin ]=[Ctotal ]+Bmax [ Actin ]+Kd−[[Ctotal ]+Bmax[Actin]+Kd)2−4Bmax[Ctotal ][ Actin ]2[ Actin ](Eq. 3) Data were fit using the Microsoft Excel Solver package by varying the values of Bmax and Kd and minimizing the sum of squares between the actual and predicted binding ratios. Significance was calculated using analysis of variance and post hoc Bonferonni t tests. Results were considered significant at p < 0.01. Analysis of Actin Cross-linking—Actin cross-linking was assessed by measurement of solution turbidity as described by Moos et al. (28Moos C. Mason C.M. Besterman J.M. Feng I.M. Dubin J.H. J. Mol. Biol. 1978; 124: 571-586Crossref PubMed Scopus (135) Google Scholar). Samples containing mixtures of recombinant cMyBP-C proteins and F-actin were prepared as described above for cosedimentation assays and allowed to equilibrate for 30 min at room temperature. Light absorbance at λ = 350 nm was measured using a Beckman DU730 spectrophotometer. Samples containing F-actin alone served as blanks. Electron Microscopy—5 μm F-actin and 30 μm recombinant proteins were mixed as described for cosedimentation assays with the exception that the centrifugation step prior to combining the proteins was omitted. Samples were incubated at room temperature for >30 min and 5-μl aliquots of each sample were applied to a freshly evaporated carbon film supported by holey formvar-covered grids. After adsorption to the carbon film (30 s), excess sample was rinsed off with 8 drops of 100 mm ammonium acetate. The ammonium acetate rinse was followed by application of 8 drops of 1% uranyl acetate for negative staining. Excess uranyl acetate was removed with filter paper after 30 s leaving a thin film of negative stain, which was then allowed to dry. Dried grids were examined and photographed at 80 kV in a JEOL-1200EXII electron microscope equipped with a 2k × 2k AMT HR60 High resolution Digital Camera (AMT, Danvers, MA). The Regulatory Motif of cMyBP-C Binds to F-actin—We sought to quantify binding interactions between the cMyBP-C N terminus and actin by measuring binding affinity between the 4 N-terminal domains of cMyBP-C (i.e. C0C2 comprised of C0-C1-m-C2 and inclusive of the proline-alanine-rich linker sequence between C0 and C1) and F-actin using high speed cosedimentation assays. C0C2 is soluble at physiological ionic strength in the absence of actin and does not sediment during high speed centrifugation. However, when combined with F-actin, C0C2 readily pellets along with actin (Fig. 1), suggesting binding interactions between the two proteins. Binding was saturable at approximately a 1:1 molar ratio of C0C2 bound to actin (Bmax = 0.92 ± 0.11) and a Kd of 13.7 ± 5.5 μm (Fig. 1B and Table 1). C1C2 (i.e. C1-m-C2, which lacks the cardiac-specific C0 domain and the proline-alanine-rich region preceding C1) also bound to actin with comparable affinity (Kd = 10.9 ± 2.2 μm and Bmax = 1.03 ± 0.08), suggesting that the C0 domain and the Pro-Ala-rich region do not significantly influence interactions with actin under these assay conditions.TABLE 1Summary data of dissociation constants (Kd) and molar binding ratios (Bmax) for binding of recombinant cMyBP-C proteins to F-actinProteinnKdBmaxμmmol/mol actinC0C2713.7 ± 5.50.92 ± 0.11C1C2 (pH 7.4)610.9 ± 2.21.03 ± 0.08C1C2 (pH 8.0)68.3 ± 3.50.65 ± 0.10aSignificant difference relative to C1C2 (p < 0.01).C1C2P713.3 ± 2.80.49 ± 0.16aSignificant difference relative to C1C2 (p < 0.01).C1m48.7 ± 2.40.92 ± 0.14C0C1740.4 ± 16.8aSignificant difference relative to C1C2 (p < 0.01).0.45 ± 0.10aSignificant difference relative to C1C2 (p < 0.01).mC2510.5 ± 4.70.59 ± 0.13aSignificant difference relative to C1C2 (p < 0.01).C3mC4511.2 ± 3.30.50 ± 0.09aSignificant difference relative to C1C2 (p < 0.01).a Significant difference relative to C1C2 (p < 0.01). Open table in a new tab To further map actin binding site(s) to specific domains of cMyBP-C, we first divided the 4 N-terminal domains of cMyBP-C into two non-overlapping recombinant fragments, C0C1 (inclusive of the Pro-Ala region) and mC2, and quantified binding of each subfragment to actin. As shown in Fig. 2, C0C1 binding to actin was significantly reduced compared with C0C2, with Kd = 40.4 ± 16.8 μm and Bmax = 0.45 ± 0.10. Although binding appeared saturable, the data could also be fit well to a straight line, suggesting that binding of C0C1 was weak and nonspecific or electrostatic in nature. By contrast, the binding affinity of mC2 (Fig. 2) was saturable with an affinity (Kd = 10.5 ± 4.7 μm) similar to that of C0C2, although the total amount of mC2 bound was reduced as shown by a decrease in Bmax (0.59 ± 0.13). To determine whether the m-domain was responsible for binding of mC2, we also investigated binding of the m-domain in combination with C1, i.e. C1m. As shown in Fig. 2, the Kd for C1m binding to actin was 8.7 ± 2.4 μm, not significantly different from that of C0C2. Bmax (0.92 ± 0.14) was also near 1.0 indicating a 1:1 binding stoichiometry comparable with C0C2. Thus, the m-domain in combination with C1 completely recapitulated the actin binding properties of the larger C0C2 fragment. Because C1m and mC2 both bound to actin with similar affinities, we next investigated whether the m-domain alone was sufficient to confer actin binding properties to a segment of cMyBP-C that does not bind actin. As shown in Fig. 3, the recombinant protein, C3C4, consisting of domains C3 and C4 of cMyBP-C, binds F-actin nonspecifically as determined by its linear actin binding relation. However, when the m-domain was inserted between domains C3 and C4 to create the C3mC4 protein, saturable binding to actin was observed. The Kd for binding was 11.2 ± 3.3 μm, not significantly different from C0C2 (Table 1). These data thus demonstrate that the regulatory m-domain of cMyBP-C binds specifically to actin and that binding is of sufficient affinity to contribute to the binding properties observed for C0C2. However, the total amount of C3mC4 bound to actin was significantly reduced compared with C0C2 (Bmax = 0.50 ± 0.09 versus 0.92 ± 0.11, p < 0.001). Multiple Actin Binding Sites of C0C2 Cross-link F-actin—A straight-forward explanation for the 1:1 molar binding stoichiometry observed for C0C2, C1C2, and C1m is that each molecule binds to a single binding site on an actin monomer. However, a different model is needed to account for the reduced binding ratios (∼1:2 actin) observed for C3mC4 or mC2. One possibility is that instead of binding to a single site on actin the C1 and m-domains bind to independent sites such that the total amount of protein bound to F-actin (1:1 for C1m, C1C2, and C0C2) reflects contributions from each of two independent sites (∼0.5 mol/mol actin for each site). To test this idea, we assessed the ability of various domains to cross-link actin filaments because cross-linking depends on the presence of multiple interaction sites with each site interacting with a different filament to form bundles or meshes (29Korn E.D. Physiol. Rev. 1982; 62: 672-737Crossref PubMed Scopus (507) Google Scholar). The extent of cross-linking can be quantified by measuring solution turbidity as assessed by light absorbance at 350 nm (28Moos C. Mason C.M. Besterman J.M. Feng I.M. Dubin J.H. J. Mol. Biol. 1978; 124: 571-586Crossref PubMed Scopus (135) Google Scholar, 30Ishikawa M. Murofushi H. Sakai H. J. Biochem. (Tokyo). 1983; 94: 1209-1217Crossref PubMed Scopus (53) Google Scholar). As shown in Fig. 4, combining C0C2 with F-actin under conditions identical to those used for cosedimentation assays significantly increased solution turbidity relative to C0C2 alone, consistent with the idea that addition of C0C2 to F-actin promotes formation of actin cross-links or bundles. C1C2 and C1m also increased turbidity, whereas C0C1, mC2, C3C4, and C3mC4 did not. Because proteins that did not increase turbidity all lacked one or both the C1 and m-domains, the results suggest that both C1 and the m-domains contain actin binding sites. To confirm that the observed increases in solution turbidity corresponded to actin bundling, electron microscopy was used to visualize F-actin filaments in the presence and absence of recombinant proteins. As shown in Fig. 5, F-actin in the absence of recombinant cMyBP-C proteins (Fig. 5A) appeared as single filaments deposited randomly on the grid surface. Punctate staining was also visible and was likely due to aggregation of actin monomers. By contrast, when combined with C0C2 or C1C2, F-actin formed thick tightly packed bundles (Fig. 5, B and C). Thick, regularly packed bundles were also readily apparent in the presence of C1m (Fig. 5D), although the extent of bundling as determined by the number and thickness of the bundles was somewhat reduced. The extent and quality of bundling was further diminished in the presence of C0C1 (Fig. 5E) or mC2 (Fig. 5F) because few bundles were observed and those that were seen were less regular and less tightly packed than those formed in the presence of C0C2, C1C2, or C1m. In particular, a regular side by side arrangement of filaments was not observed in the presence of C0C1. Instead, filaments appeared only loosely connected (Fig. 5H). This infrequent, loose association was most likely the result of nonspecific accumulation of filaments around protein aggregates rather than from true actin cross-linking. The bundles formed by mC2 (Fig. 5I) appeared somewhat better organized than those formed in the presence of C0C1, but their thickness and frequency were still significantly reduced compared with C0C2, C1C2, or C1m. Thus, tight, regularly packed bundles indicative of actin cross-linking were most commonly observed in the presence of C0C2, C1C2, and C1m, a result in good agreement with solution turbidity measurements. Although it is unlikely that the C1 and m-domain promote actin bundling in vivo because of reduced protein stoichiometry of cMyBP-C to actin in sarcomeres, the presence of regulatory proteins, and other constraints on sarcomere geometry, the results provide evidence for multiple