Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations

Fiona L. Wong, Thomas A. Bunch, Victoria C. Lepak, Allison L. Steedman, Brett A. Colson

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

N-terminal cardiac myosin-binding protein C (cMyBP-C) domains (C0-C2) bind to thick (myosin) and thin (actin) filaments to coordinate contraction and relaxation of the heart. These interactions are regulated by phosphorylation of the M-domain situated between domains C1 and C2. In cardiomyopathies and heart failure, phosphorylation of cMyBP-C is significantly altered. We aimed to investigate how cMyBP-C interacts with myosin and actin. We developed complementary, high-throughput, C0-C2 FRET-based binding assays for myosin and actin to characterize the effects due to 5 HCM-linked variants or functional mutations in unphosphorylated and phosphorylated C0-C2. The assays indicated that phosphorylation decreases binding to both myosin and actin, whereas the HCM mutations in M-domain generally increase binding. The effects of mutations were greatest in phosphorylated C0-C2, and some mutations had a larger effect on actin than myosin binding. Phosphorylation also altered the spatial relationship of the probes on C0-C2 and actin. The magnitude of these structural changes was dependent on C0-C2 probe location (C0, C1, or M-domain). We conclude that binding can differ between myosin and actin due to phosphorylation or mutations. Additionally, these variables can change the mode of binding, affecting which of the interactions in cMyBP-C N-terminal domains with myosin or actin take place. The opposite effects of phosphorylation and M-domain mutations is consistent with the idea that cMyBP-C phosphorylation is critical for normal cardiac function. The precision of these assays is indicative of their usefulness in high-throughput screening of drug libraries for targeting cMyBP-C as therapy.

Original languageEnglish (US)
Pages (from-to)125-137
Number of pages13
JournalJournal of Molecular and Cellular Cardiology
Volume186
DOIs
StatePublished - Jan 2024

Keywords

  • Actomyosin
  • Cardiac myosin-binding protein C (cMyBP-C)
  • Fluorescence resonance energy transfer (FRET)
  • High-throughput screen (HTS)
  • Hypertrophic cardiomyopathy (HCM)
  • Protein kinase a (PKA) regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Fingerprint

Dive into the research topics of 'Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations'. Together they form a unique fingerprint.

Cite this