Microscale thermophoresis suggests a new model of regulation of cardiac myosin function via interaction with cardiac myosin-binding protein C

Figures

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  Figure 1. Myosin filament–based regulation in the heart.A, cartoon representation of the myosin filament OFF (left) and ON state (right). The myosin catalytic domain (CD), essential light chain (ELC), and regulatory light chain (RLC) are shown red, green, and blue, respectively. The positions of M-band and Z-disc are indicated accordingly. B, atomic model of cardiac myosin in the interacting heads motif (Protein Data Bank: 5TBY). Blocked head and free head and myosin tails (S2Δ) are labeled accordingly. C, normalized MST binding curves for myosin S2Δ titrated against Alexa647-labeled myosin S1 (red) or myosin S2Δ (blue). Means ± SEM, n = 8. MST, microscale thermophoresis.

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  Figure 2. Interaction of N-terminal domains of cMyBP-C with cardiac myosin.A, domain structure and known interaction partners of cMyBP-C. The P/A linker and m-motif (m) are labeled accordingly. B, left, SDS-PAGE and structure of isolated myosin S1 with myosin heavy chain (MHC), essential light chain (ELC), and regulatory light chain (RLC) labeled accordingly. Right, normalized MST binding curves for different cMyBP-C fragments titrated against myosin S1. C, left, SDS-PAGE and structure of miniHMM with myosin tail part (myosin S2Δ) and RLC labeled accordingly. Right, normalized MST binding curves for different cMyBP-C fragments titrated against miniHMM. D, left, SDS-PAGE and structure of myosin S2Δ. Right, normalized MST binding curves for different cMyBP-C fragments titrated against myosin S2Δ. Means ± SEM, n = 3 to 6. cMyBP-C, cardiac isoform of myosin-binding protein C; MST, microscale thermophoresis; P/A, proline- and alanine-rich.

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  Figure 3. Effect of phosphorylation on C0C2–cardiac myosin interactions.A, Phostag-SDS-PAGE of C0C2 before (−) and after in vitro phosphorylation with PKA (+). Individual phosphospecies are clearly separated by Phostag-SDS-PAGE and labeled accordingly. B, normalized MST binding curves for unphosphorylated (C0C2, black) and tris-phosphorylated C0C2 (C0C2-3P, red) titrated against cardiac myosin S1, miniHMM, and myosin S2Δ. C, left, in vitro phosphorylation of miniHMM by cMLCK resolved by Phostag-SDS-PAGE. Right, normalized MST binding curves for C0C2 (black) and C0C2-3P (red) titrated against phospho-miniHMM. D, cartoon representation for the effects of C0C2 phosphorylation on binding to myosin. See main text for details. Means ± SEM, n = 3 to 4. Statistical significance of differences between C0C2 and C0C2-3P was assessed with an unpaired two-tailed Student's t test: ∗∗p < 0.01, ∗p < 0.05, ns. CD, catalytic domain; cMLCK, cardiac isoform of myosin light chain kinase; C0C2, cMyBP-C spanning domains C0 to C2; ELC, essential light chain; ns, not significant; RLC, regulatory light chain.

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  Figure 4. The central domains of cMyBP-C tightly bind to the myosin heads.A, left, SDS-PAGE of purified full length (FL) and C0C2-truncated cMyBP-C (ΔC0C2). Right, normalized MST binding curves for full length (FL) (black) and ΔC0C2–cMyBP-C (red) titrated against myosin S1. B, normalized MST binding curves for C2C4 titrated against myosin S1 (red), myosin S2Δ (blue), and miniHMM (green). C, normalized MST binding curves for C5C7 titrated against myosin S1 (red), myosin S2Δ (blue) and miniHMM (green). D, left, competition model for C2C4 and C5C7 binding to the same site on myosin S1. Right, competitive titration of C5C7 into a mixture of myosin S1 and C2C4. The binding curves for C5C7 in the absence and presence of C2C4 are shown in purple and blue, respectively. The simulated data from the model are shown in the plot as a black dashed line. E, effect of the HCM-associated N755K (blue), R820Q (purple), and R943Q (red) substitutions in C5C7 on binding to myosin S1 (left) and myosin S2Δ (right). Means ± SEM, n = 3 to 4. Statistical significance of differences versus wildtype (WT) was assessed with a one-way ANOVA followed by Dunnett's multiple comparison: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <…

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  Figure 5. Hypothetical model for the role of cMyBP-C during cardiac muscle activation.A, in the dephosphorylated state, cMyBP-C (dark gray) is bound to myosin catalytic domains of the myosin blocked head (red) and free head (purple) via interactions of its central domains (C2C4 and C5C7, respectively) and C1/m-motif with the RLC–neck region. The N-terminal domain C0 is in a conformational equilibrium between thin filament bound and unbound states, determined by the flexibility and elongation of the P/A linker. B, phosphorylation (yellow circles) of cMyBP-C abolishes the m-motif–myosin S2 interaction, which releases parts of cMyBP-C (and associated myosin heads) from the thick filament backbone, and increases the probability of C0–thin filament interactions. C, calcium activation of the thin filaments subsequently leads to cMyBP-C domain C1 cooperatively binding to available actin-binding sites in proximity to C0, which would fully activate the thin filament. In addition, this structural rearrangement would bring cMyBP-C–attached myosin heads closer to the surface of the calcium-activated thin filament and increase the probability of actomyosin crossbridge formation. cMyBP-C, cardiac…