Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET. (May 2022)

• Record Type: Journal Article
• Authors: Kanassatega, Rhye-Samuel; Bunch, Thomas A.; Lepak, Victoria C.; Wang, Christopher; Colson, Brett A.
• Is Part Of: Journal of molecular and cellular cardiology. Volume 166(2022)
• Page Start: 116
• Page End: 126
• Publication Date: 2022-05
• Subjects: Cardiac myosin-binding protein C (cMyBP-C) -- Biosensor -- Phosphorylation -- Protein kinase A (PKA) -- Fluorescence lifetime -- High-throughput screening (HTS) -- Time-resolved fluorescence resonance energy transfer (TR-FRET); cMyBP-C Cardiac myosin-binding protein C -- C0-C2 cMyBP-C N-terminal domains/portion C0 through C2 -- TR-FRET time-resolved fluorescence resonance energy transfer -- HCM hypertrophic cardiomyopathy -- HF heart failure -- S2 myosin subfragment-2 -- RLC myosin regulatory light chain -- P/A proline/alanine-rich linker -- Tm tropomyosin -- cTnI cardiac troponin-I -- M-domain motif domain -- IAEDANS (5-((((2-Iodoacetyl)amino)ethyl)amino)Naphthalene-1-Sulfonic acid -- DDPM N-(4-(dimethylamino)-3, 5-dinitrophenyl)maleimide -- FLTPR fluorescence lifetime plate reader -- HTS high-throughput screening -- TCEP tris(2-carboxyethyl)phosphine -- DTT dithiothreitol -- CD circular dichroism -- IRF instrument response function -- Donly donor-only -- D-A donor-acceptor -- FWHM full width at half-maximum -- MRE molar residue ellipticity -- C0-C2Cys225.Cys330 biosensor; Cardiology -- Periodicals; Heart Diseases -- Periodicals; Molecular Biology -- Periodicals; Cardiologie -- Périodiques; Cardiology; Electronic journals; Periodicals; 616.12
• DOI: 10.1016/j.yjmcc.2022.02.005
• Languages: English
• ISSNs: 0022-2828

Abstract

Abstract: 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. Graphical abstract: Unlabelled Image Highlights: 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.