Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET

• Published in: Journal of molecular and cellular cardiology Vol. 166; pp. 116 - 126
• Main Authors: Kanassatega, Rhye-Samuel, Bunch, Thomas A., Lepak, Victoria C., Wang, Christopher, Colson, Brett A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.05.2022
• Subjects: Biosensor; Cardiac myosin-binding protein C (cMyBP-C); Carrier Proteins; Fluorescence lifetime; Fluorescence Resonance Energy Transfer; Heart Failure - genetics; Heart Failure - metabolism; High-throughput screening (HTS); Humans; Mutation; Phosphorylation; Protein kinase A (PKA); Time-resolved fluorescence resonance energy transfer (TR-FRET); MRE; Phosphorylation; cTnI; Donly; P/A; cMyBP-C; DTT; HTS; RLC; Cardiac myosin-binding protein C (cMyBP-C); C0-C2; IAEDANS; C0-C2Cys225.Cys330; High-throughput screening (HTS); Tm; Fluorescence lifetime; CD; M-domain; DDPM; FLTPR; HCM; Biosensor; FWHM; D-A; Protein kinase A (PKA); IRF; Time-resolved fluorescence resonance energy transfer (TR-FRET); TCEP; TR-FRET; HF; S2
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2022.02.005

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating contractile dysfunction in heart failure. Mimicking the structural dynamics of phosphorylated cMyBP-C by small-molecule drug binding could lead to therapies that modulate cMyBP-C conformational states, and thereby function, to improve contractility. We have developed a human cMyBP-C biosensor capable of detecting intramolecular structural changes due to phosphorylation and mutation. Using site-directed mutagenesis and time-resolved fluorescence resonance energy transfer (TR-FRET), we substituted cysteines in cMyBP-C N-terminal domains C0 through C2 (C0-C2) for thiol-reactive fluorescent probe labeling to examine C0-C2 structure. We identified a cysteine pair that upon donor-acceptor labeling reports phosphorylation-sensitive structural changes between the C1 domain and the tri-helix bundle of the M-domain that links C1 to C2. Phosphorylation reduced FRET efficiency by ~18%, corresponding to a ~11% increase in the distance between probes and a ~30% increase in disorder between them. The magnitude and precision of phosphorylation-mediated TR-FRET changes, as quantified by the Z'-factor, demonstrate the assay's potential for structure-based high-throughput screening of compounds for cMyBP-C-targeted therapies to improve cardiac performance in heart failure. Additionally, by probing C1's spatial positioning relative to the tri-helix bundle, these findings provide new molecular insight into the structural dynamics of phosphoregulation as well as mutations in cMyBP-C. Biosensor sensitivity to disease-relevant mutations in C0-C2 was demonstrated by examination of the hypertrophic cardiomyopathy mutation R282W. The results presented here support a screening platform to identify small molecules that regulate N-terminal cMyBP-C conformational states. [Display omitted] •Phosphorylation governs cMyBP-C binding to actomyosin by key structural changes.•We engineered cysteine probe pairs in cMyBP-C's N-terminus for time-resolved FRET.•Phosphorylation and mutation of cMyBP-C caused changes in FRET Efficiency of probes.•Sites in C1 and M -domains important for binding showed the largest FRET changes.•Precise cMyBP-C FRET assays are excellent tools for high-throughput drug discovery.