Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85724, United States of America.
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85724, United States of America.
J Mol Cell Cardiol. 2022 May;166:116-126. doi: 10.1016/j.yjmcc.2022.02.005. Epub 2022 Feb 25.
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.
心肌肌球蛋白结合蛋白 C（cMyBP-C）是肌节中粗丝相关蛋白，也是治疗心力衰竭收缩功能障碍的潜在治疗靶点。通过小分子药物结合模拟磷酸化 cMyBP-C 的结构动力学，可能会产生调节 cMyBP-C 构象状态和功能从而改善收缩力的治疗方法。我们开发了一种能够检测由于磷酸化和突变引起的分子内结构变化的人源 cMyBP-C 生物传感器。我们使用定点突变和时间分辨荧光共振能量转移（TR-FRET），将 cMyBP-C N 端结构域 C0 到 C2（C0-C2）中的半胱氨酸替换为硫醇反应性荧光探针标记，以研究 C0-C2 结构。我们确定了一对半胱氨酸，在供体-受体标记后，报告 C1 结构域和连接 C1 到 C2 的 M 结构域三螺旋束之间的磷酸化敏感结构变化。磷酸化使 FRET 效率降低了约 18%，对应于探针之间距离增加约 11%，无序度增加约 30%。通过 Z'-因子定量的磷酸化介导的 TR-FRET 变化的幅度和精度表明，该测定法具有基于结构的高通量筛选化合物的潜力，用于针对 cMyBP-C 的治疗以改善心力衰竭中的心脏性能。此外，通过探测 C1 相对于三螺旋束的空间定位，这些发现为磷酸化调节以及 cMyBP-C 突变的结构动力学提供了新的分子见解。通过检查肥厚型心肌病突变 R282W，证明了生物传感器对 C0-C2 中与疾病相关的突变的敏感性。这里呈现的结果支持了一种筛选平台，用于识别调节 N 端 cMyBP-C 构象状态的小分子。
