Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

Abstract

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Fig. 1.

(A) Actin filament twisting motions in the μs time range are detected by TPA. (B) Domain organization of MyBP-C isoforms. Domains listed from N-terminal C0 to C-terminal C10, including the Pro/Ala rich linker (L_P/A) and phosphorylation motif (m). (C) Effect of 1 μM fast skeletal (red), 1 μM slow skeletal (blue) or 4 μM cMyBP-C (black) on the TPA decay of ErIA-actin (gray). (D) Dependence of final anisotropy on actin binding.

Fig. 2.

(A) Final anisotropy of actin (Fig. 1) as a function of the fraction bound to isoforms of MyBP-C. Effects in the absence (white bars) and presence (cyan bars) of PKA-mediated phosphorylation on the angular amplitudes (B, Eq. 3) and rates (C, Eq. 4) of actin torsional dynamics (Fig. 1A) at saturation. *Significant change in absence of PKA treatment compared to actin alone (P < 0.05). ^#Significant change due to PKA treatment (P < 0.05).

Fig. 3.

Effect of cMyBP-C fragments (A) and PKA treatment on actin twisting dynamics detected by TPA. Effects in the absence (white bars) and presence (cyan bars) of PKA treatment on the amplitudes (B, Eq. 3) and rates (C, Eq. 4) of actin torsional dynamics at saturation. *Significant change in absence of PKA treatment compared to actin alone (P < 0.05). ^#Significant change due to PKA treatment (P < 0.05). Full-length cMyBP-C (FL) shown in Fig. 1B.