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

Inchingolo, Alessio Vincenzo, Previs, Samantha Beck, Previs, Michael J., Warshaw, David M., Kad, Neil M (2019) Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding. Proceedings of the National Academy of Sciences, . ISSN 0027-8424. (doi:10.1073/pnas.1816480116)

Abstract

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.

Item Type: Article
DOI/Identification number: 10.1073/pnas.1816480116
Uncontrolled keywords: cardiomyopathy, muscle, contractility, single molecule, regulated thin filaments
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: N. Kad
Date Deposited: 26 Mar 2019 11:17 UTC
Last Modified: 03 Jun 2019 09:36 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/73203 (The current URI for this page, for reference purposes)
Kad, Neil M: https://orcid.org/0000-0002-3491-8595
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