Structural and biochemical investigations into the mechanism and evolution of soluble guanylate cyclase regulation

项目总结 一氧化氮(NO)是一种细胞间信号分子，在生命的几个王国中使用，在动物中， NO参与血管扩张、神经传递和宿主对感染的反应等关键过程。中环 在动物生理学中起作用的是金属酶可溶性鸟苷环化酶(SGC)，它产生 次要信使3‘，5’-一磷酸鸟苷(CGMP)对NO1，2的反应 对于人类的几个生理过程，sGC是目前的治疗靶点3，了解其结构- SGC激活的基础将为继续发展提供信息。尽管不只被证明是 结合在血红素辅因子上，sGC活性有三个不同的阶段：1)基础活性低，无结合(NO-1)； 游离)，2)中等活性(增加10倍)，具有单一的血红素结合的NO(1-NO)，以及3)最大活性(200- 倍数增加)，剩余的4号。生物化学5和结构6，7实验表明没有变构调节 通过延长N端无结合调节结构域之间的盘绕结构域激活SGC 和C末端催化结构域。然而，结构域之间的排列和构象人口分布是不同的。 生理上相关的1-NO状态的作用仍有待充分探索。而后生动物SGCs则以 只有一个血红素结合区的异源二聚体--新近发现的单细胞真核生物凤尾藻 FlexA(CF)sGC是一种同源二聚体，但仍显示出半个位点的血红素负载和3个阶段的无激活8，9。 通过这项拟议的研究，我们将探索CF sGC如何通过变构调节活性来响应NO 刺激。活性分析、同源引导的突变研究和小分子刺激物的调查将 可用于表征CFsGC的活性。将使用低温电子显微镜来确定高分辨率 活性和非活性状态下的CF sGC的结构，允许可视化关键的结构相似性和 CFsGC与动物sGC之间的差异。小角X射线散射(SAXS)实验将 使用CFsGC和动物sGCs进行去卷积，推测sGC的3阶段无激活图谱。 这些实验旨在识别和描述CFsGC的结构状态，并阐明其结构 祖先和动物sGC之间的差异，定义了基于NO的信号的进化史。

Project summary Nitric oxide (NO) is an intercellular signaling molecule used across several kingdoms of life, and in animals, NO is involved in key processes like vasodilation, neurotransmission, and the host response to infection. Central to NO function in animal physiology is the metalloenzyme soluble guanylate cyclase (sGC), which generates the secondary messenger guanosine 3’,5’-monophosphate (cGMP) in response to NO1,2. Because of its centrality to several physiological processes in humans, sGC is a current therapeutic target3, and understanding the struc- tural underpinnings of sGC activation will inform continued development. Though NO has only been proven to bind at the heme cofactor, sGC activity has three distinct stages: 1) low basal activity without NO bound (NO- free), 2) moderate activity (10-fold increase) with a single, heme-bound NO (1-NO), and 3) maximal activity (200- fold increase) with excess NO4. Biochemical5 and structural6,7 experiments suggest NO allosterically regulates sGC activation through elongation of a coiled-coil domain between the N-terminal NO-binding regulatory domain and the C-terminal catalytic domain. However, interdomain arrangement and the conformational population dis- tribution of the physiologically relevant 1-NO state remain to be fully explored. While metazoan sGCs exist as heterodimers with only one heme-binding domain, the recently discovered single-celled eukaryotic Choanoeca flexa (Cf) sGC is a homodimer that nonetheless exhibits half-sites heme-loading and 3-stage NO-activation8,9. Through this proposed research, we will probe how Cf sGC allosterically modulates activity in response to NO stimulation. Activity assays, homology-guided mutational studies, and a survey of small molecule stimulators will be used to characterize Cf sGC activation. Cryo-electron microscopy will be used to determine high-resolution structures of Cf sGC in active and inactive states, allowing for visualization of key structural similarities and differences between Cf sGC and animal counterparts. Small-angle X-ray scattering (SAXS) experiments will be performed with both Cf sGC and animal sGCs to deconvolve the putative 3-stage NO-activation profile of sGC. These experiments aim to identify and describe the structural states of Cf sGC and illuminate the structural differences between ancestral and animal sGCs that define the evolutionary history of NO-based signaling.