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Abstract
Cardiac myosin–binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0–C2) to labeled F-actin reduced rotational flexibility by 20–25°, indicated by increased final anisotropy of the TPA decay. The effects of C0–C2 on actin TPA were highly cooperative (n = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0–C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser to Asp phosphomimetic substitutions in the M-domain of C0–C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0–C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0–C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3β) site in the Pro/Ala-rich linker of C0–C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C–induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin-myosin interactions.
Highlights
Cardiac myosin– binding protein C is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner
Thick and thin filament structure and contractile function are further regulated by cardiac myosin– binding protein C. cMyBP-C is a modular thick filament-associated protein located in the C-zone, where actinmyosin crossbridges are found [5]
To determine the ratios of cMyBP-C to actin in the transient phosphorescence anisotropy (TPA) experiments, we measured binding of N-terminal cMyBP-C variants to 1 M phalloidin-stabilized ErIA-actin by cosedimentation under the same conditions as in our TPA experiments
Summary
Cardiac myosin– binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. Binding of cMyBP-C N-terminal domains (C0 –C2) to labeled F-actin reduced rotational flexibility by 20 –25°, indicated by increased final anisotropy of the TPA decay. The effects of C0 –C2 on actin TPA were highly cooperative (n ؍ϳ8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). C0 –C1 (lacking M-domain/C2) exhibited reduced cooperativity, but not as reduced as by phosphorylated C0 –C2 These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. PKA-mediated phosphorylation of cMyBP-C modulates the kinetics of actin-myosin crossbridge cycling by reducing binding to actin [9] or myosin subfragment 2 [10]. The N-terminal domains C0 –C2 binding to filamentous actin has been been extensively studied by cosedimentation assays [9, 14], small-angle X-ray/neutron scattering [15, 16], and laser
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