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 (K(d)) of approximately 10 microm and a molar binding ratio (B(max)) 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 B(max)) 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.

FIGURE 1.

Cosedimentation binding data for C0C2 and C1C2. A, representative high speed cosedimentation binding experiment. C0C2 (1-30 μm final concentrations) was mixed with 5 μm F-actin (final concentration) and spun for 30 min at 390,000 × g. The amount of C0C2 and actin in pellet fractions (left) was quantified by SDS-PAGE using a standard curve run on each gel with varying known molar ratios of C0C2 to actin (right). B, summary data (means ± S.D.) for C0C2 and C1C2 binding to actin. The K_d and B_max values for C0C2 (circles) were 13.7 ± 5.5 μm and 0.92 ± 0.11 mol/mol (n = 7). K_d and B_max values for C1C2 (squares) were 10.9 ± 2.2 μm and 1.03 ± 0.08 mol/mol (n = 6).

FIGURE 2.

Summary cosedimentation binding data for C1m, mC2, and C0C1. C1m (circles), K_d = 8.7 ± 2.4 μm, B_max = 0.92 ± 0.14 (n = 4); mC2 (squares), K_d = 10.5 ± 4.7 μm, B_max = 0.59 ± 0.13 (n = 5); C0C1 (triangles), K_d = 40.4 ± 16.8 μm, B_max = 0.45 ± 0.10 (n = 7). Data are means ± S.D.

FIGURE 3.

The MyBP-C m-domain binds specifically to actin. Cosedimentation binding data (means ± S.D.) for C3C4 (circles) comprised of domains C3 and C4 of cMyBP-C and for C3mC4 (squares) an engineered protein in which the m-domain sequence was inserted between domains 3 and 4 of cMyBP-C. C3C4 bound nonspecifically to actin as shown by the linear binding relation, whereas C3mC4 bound to actin in a saturable manner with K_d = 11.2 ± 3.3 μm, B_max = 0.50 ± 0.09 (n = 5).

FIGURE 4.

Bundling of F-actin by C1 and the m-domain. F-actin was combined with recombinant cMyBP-C proteins under conditions identical to those used for cosedimentation binding assays (see “Experimental Procedures”) and solution turbidity was measured as light absorbance at λ = 350 nm. Proteins containing both C1 and the m-domain (i.e. C0C2, C1C2, and C1m) increased solution turbidity in a concentration-dependent manner, whereas C0C1, mC2, C3C4, and C3mC4 did not increase solution turbidity. Data are means ± S.D. A.U., arbitrary units.

FIGURE 5.

Electron micrographs of F-actin in the presence of recombinant cMyBP-C proteins. 5 μm F-actin was combined with 30 μm recombinant cMyBP-C proteins (1:6 ratio) and processed as described under “Experimental Proceduress” for visualization by electron microscopy. Scale bars represent 200 nm in A-F and 50 nm in G-I. A, F-actin alone; B, F-actin plus C0C2; C, F-actin plus C1C2; D, F-actin plus C1m; E, F-actin plus C0C1; F, F-actin plus mC2. Thick bundles of F-actin are apparent in the presence of C0C2 (B), C1C2 (C), and C1m (D), but are either not present in the absence of added proteins (A, F-actin alone) or reduced in the presence of C0C1 (E) or mC2 (F). Panels G-I show higher magnification images of F-actin bundles in the presence of C1m (G), C0C1 (H), and mC2 (I) to illustrate tighter more regular packing of F-actin in the presence of C1m relative to either C0C1 or mC2.

FIGURE 6.

Effects of phosphorylation of the m-domain and alkaline pH on C1C2 binding to actin. A, cosedimentation binding data (means ± S.D.) for phosphorylated C1C2 (i.e. C1C2P, filled circles). Pro Q Diamond staining (inset) confirmed phosphorylation of C1C2P following treatment with PKA. C1C2P bound to F-actin with K_d = 13.3 ± 2.8 μm, B_max = 0.49 ± 0.16 (n = 7). Binding data for C1C2 (open circles) were redrawn from Fig. 1 for comparison. B, PKA treatment of C1C2 abolished the ability of C1C2P to increase the turbidity of F-actin solutions. C, cosedimentation binding data for C1C2 collected at pH 8.0 (closed circles), K_d = 8.3 ± 3.5 μm, B_max = 0.65 ± 0.10 (n = 6). Binding data for C1C2 at pH 7.4 (open circles), were redrawn from Fig. 1 for comparison. D, turbidity of solutions containing F-actin and C1C2 was reduced at pH 8.0. A.U., arbitrary units.

FIGURE 7.

Cosedimentation binding of C1C2 to F-actin in the presence of S2Δ. A, cosedimentation binding experiments with 5 μm F-actin performed in the presence of 30 μm S2Δ. The presence of S2Δ did not significantly affect binding of C1C2 to F-actin (K_d = 15.4 ± 3.0 μm and B_max = 0.97 ± 0.10, n = 4). B, cosedimentation binding assays performed with 5 μm F-actin and 10 μm C1C2 in the presence of 1-50 μm S2Δ. The amount of C1C2 (circles) and S2Δ (squares) found in the pellets did not change in the presence of increasing amounts of S2Δ. Data are means ± S.D.

FIGURE 8.

Cosedimentation binding of C1C2 to native thin filaments. NTF were prepared as described under “Experimental Procedures” from bovine heart and 5 μm purified NTF were used in cosedimentation binding assays. An SDS-PAGE of purified NTF is shown in the inset. Positions of actin, tropomyosin (Tm), troponin I (TnI), and troponin T (TnT) are indicated. Troponin C is not visible on the gel due to weak staining by Coomassie Blue. C1C2 bound to NTF in a saturable manner in the presence (open circles) or absence (closed circle) of Ca²⁺ (means ± S.D.). K_d and B_max values were 8.4 ± 4.6 and 0.90 ± 0.13 μm (n = 6) for C1C2 in the presence of Ca²⁺ and 12.5 ± 4.8 and 0.90 ± 0.13 μm (n = 5) in the absence of Ca²⁺. Solutions lacking Ca²⁺ contained (in mmol/liter): 180 KCl, 20 imidazole, pH 7.4, 1 EGTA, 1 MgCl₂, and 1 DTT. Solutions with Ca²⁺ contained additional CaCl₂ to achieve 1 mm free Ca²⁺.

FIGURE 9.

Model of cMyBP-C interactions with F-actin. cMyBP-C (green) is depicted as a series of 12 spherical domains representing domains C0 through C10 and the MyBP-C motif or m-domain (M) between domains C1 and C2. The Pro-Ala-rich region between the C0 and C1 domains is represented as a yellow rectangle. A, the N terminus of cMyBP-C interacts with the thin filament primarily through binding of the C1 domain and the m-domain as demonstrated by data in this study. The cardiac-specific C0 domain is also shown binding to actin based on data from this and other studies (16, 19). B, binding of the M-domain to the thin filament is reduced by phosphorylation of the m-domain or by increasing pH (reduced net positive charge of the m-domain).