Mechanisms of Thin Filament Regulation by Myosin Binding Protein-C

摘要 心肌肌球蛋白结合蛋白-C（cMyBP-C）是心脏功能的重要调节因子， 对于正常的收缩和舒张心脏功能以及对于增强的收缩力响应于正性肌力是必需的。 通过多种信号传导途径磷酸化cMyBP-C的激动剂。而磷酸化是 与心脏保护相关的低磷酸化在心力衰竭中一直存在。突变 编码cMyBP-C的基因MYBPC 3也是肥厚型心肌病最常见的病因 (HCM)这种疾病的患病率约为1：500。然而，尽管它对心脏健康和疾病具有重要意义， 关于cMyBP-C如何介导其功能效应或cMyBP-C如何介导其功能效应， 磷酸化增加心脏对变力性刺激的收缩力。我们三个实验室的工作表明 明确地说，cMyBP-C直接激活肌肉肌节中的细丝， 肌球蛋白跨桥因此，我们的数据表明cMyBP-C的作用，以增加收缩或延迟放松 是由于cMyBP-C将原肌球蛋白转变为细丝上的活化状态的直接作用。这里我们 利用cryo-EM的革命来揭示cMyBP-C与薄 丝，也与肌球蛋白-S1的第一次。我们将使用冷冻电镜重建来识别关键残留物 介导与细丝和肌球蛋白-S1的相互作用，并将使用一系列 功能性方法，包括我们实验室专门开发的一种新方法（“Spy-C”）， 原位替换肌节中cMyBP-C的N ′-末端结构域。利用我们的结构图，我们证明了 通过鉴定负责原肌球蛋白的cMyBP-C活化的氨基酸来证明原理，并表明 磷酸化导致cMyBP-C结构域在细丝上重新定位。具体目标将建立在这些基础之上 发现和联合收割机我们独特的资源，以：1）确定如何个别cMyBP-C域 当与细丝和肌球蛋白-S1结合时，与相邻结构域进行通信，以及2）定义如何 Ca 2+和磷酸化调节cMyBP-C与细丝和肌球蛋白的关键调节相互作用。 S1.通过鉴定控制cMyBP-C与细丝结合的关键分子相互作用， 这些研究将为未来调节心脏收缩的靶向方法铺平道路， 放松.

ABSTRACT Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart function that is necessary for normal systolic and diastolic heart function and for enhanced contractility in response to inotropic agonists that phosphorylate cMyBP-C through numerous signaling pathways. Whereas phosphorylation is associated with cardiac protection, hypo-phosphorylation is consistently found in heart failure. Mutations in MYBPC3, the gene encoding cMyBP-C, are also the most common cause of hypertrophic cardiomyopathy (HCM), a disease with a prevalence of ~1:500. However, despite its significance to cardiac health and disease, there is still remarkably little known regarding how cMyBP-C mediates its functional effects or how cMyBP-C phosphorylation increases cardiac contractility in response to inotropic stimuli. Work from our 3 labs has shown unequivocally that cMyBP-C directly activates the thin filament in muscle sarcomeres in the same way as myosin cross-bridges. Our data thus demonstrate cMyBP-C effects to augment contraction or delay relaxation are due to direct effects of cMyBP-C to shift tropomyosin to an activated state on the thin filament. Here we take advantage of the revolution in cryo-EM to reveal detailed structures of cMyBP-C interactions with the thin filament and also with myosin-S1 for the first time. We will use cryo-EM reconstructions to identify key residues that mediate interactions with thin filaments and myosin-S1 and will test model predictions using an arsenal of functional approaches including a novel (“Spy-C”) method developed exclusively in our labs that allow us to replace the N'-terminal domains of cMyBP-C in sarcomeres in situ. Using our structural maps, we demonstrate proof of principle by identifying amino acids responsible for cMyBP-C activation of tropomyosin and show that phosphorylation causes cMyBP-C domains to reposition on the thin filament. Specific aims will build on these discoveries and combine our unique resources to: 1) Determine how individual cMyBP-C domains communicate with neighboring domains when bound to the thin filament and myosin-S1, and 2) Define how Ca2+ and phosphorylation modulate key regulatory interactions of cMyBP-C with the thin filament and myosin- S1. By identifying the critical molecular interactions that control cMyBP-C binding to the thin filament and myosin-S1 these studies will pave the way for future targeted approaches to modulate cardiac contraction and relaxation.