Phosphorylation and calcium antagonistically tune myosin-binding protein C\u27s structure and function

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C\u27s N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain\u27s extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C\u27s inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C\u27s calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C\u27s phosphorylation state

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism

Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling

The beating heart exhibits remarkable contractile fidelity over a lifetime, which reflects the tight coupling of electrical, chemical, and mechanical elements within the sarcomere, the elementary contractile unit. On a beat-to-beat basis, calcium is released from the ends of the sarcomere and must diffuse toward the sarcomere center to fully activate the myosin- and actin-based contractile proteins. The resultant spatial and temporal gradient in free calcium across the sarcomere should lead to nonuniform and inefficient activation of contraction. We show that myosin-binding protein C (MyBP-C), through its positioning on the myosin thick filaments, corrects this nonuniformity in calcium activation by exquisitely sensitizing the contractile apparatus to calcium in a manner that precisely counterbalances the calcium gradient. Thus, the presence and correct localization of MyBP-C within the sarcomere is critically important for normal cardiac function, and any disturbance of MyBP-C localization or function will contribute to the consequent cardiac pathologies

Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding

Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility

Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding

Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility