Activation and Inactivation Mechanisms of Membrane-bound Guanylyl Cyclases

生命形式是如何感知环境的？在细胞水平上，g蛋白偶联受体（gpcr）和膜结合的胍基环化酶（mGCs）在检测细胞外信号并将其传递到细胞内发挥关键作用。与GPCRs不同的是，GPCRs中有多个蛋白参与信号转导、-调节和-扩增，而mGCs将受体、调节剂和效应域组合在一个蛋白中。与gpcr不同，全长mGCs没有高分辨率的结构数据。因此，我们仍然不知道允许mgc执行这三个任务的分子机制。因此，本课题的核心课题是利用单粒子冷冻电镜（cryo-EM）确定全长mGC的三维结构，并了解其激活和失活的分子机制。这个项目的显著特点是我将充分利用海胆精子作为模型系统：在这个系统中，我们不仅对mGC的信号转导有很好的生化理解。海胆精子必须能够追踪化学引诱剂的单个分子才能找到卵子，这一事实导致了鞭毛的进化，鞭毛密集地挤满了mGC受体。我的计划是用刺海胆的精子分离出足够的材料，用低温电子显微镜和单粒子重建来确定结构。我还将诱导点状麻mGC的明确功能状态，并确定其结构，以揭示其激活和失活的分子机制。具体来说，我计划揭示化学引诱剂反应是如何以及在何种化学计量中被mGC复合物结合的；研究哪些结构重排是由细胞外区域的配体结合引起的；了解细胞外和细胞内结构域在结构和功能上是如何耦合的；揭示mGC通过磷酸化和去磷酸化调控的结构和功能方面。系统发育比较和保守的磷酸化位点表明，A. punctulata mGC是哺乳动物肽结合GC-A和GC-B的同源物。因此，这些数据不仅有助于我们对马斑金针虫精子细胞中化学引诱剂感知的分子机制有更深入的了解，而且有助于揭示真核生物中以mgc为基础的激素信号转导的分子机制。

How do life forms sense their environment? On the cellular level, both G-protein coupled receptors (GPCRs) and membrane-bound guanylyl cyclases (mGCs) play key roles in detecting extracellular signals, and relaying them to the inside of the cell. Different from GPCRs, where multiple proteins are involved in signal transduction, -modulation and -amplification, mGCs have receptor, modulator and effector domains combined into a single protein. Different from GPCRs, no high-resolution structural data is available for full-length mGCs. Consequently, we still do not know the molecular mechanisms that allow mGCs to carry out all three tasks. Core subject of this project proposal is thus determining the 3D structure of a full-length mGC using single-particle cryo-electron microscopy (cryo-EM), and gaining understanding of the molecular mechanisms of its activation and inactivation. Distinguishing feature of the project is that I will make full use of sea urchin sperm as a model system: not only do we have good biochemical understanding of signal transduction by mGC in this system. The fact that sea urchin sperm must be able to trace individual molecules of chemo-attractants to find an egg has resulted in the evolution of flagella that are densely packed with the receptor mGC. My plan is to use sperm of the sea urchin Arbacia punctulata to isolate enough material for structure determination by cryo-EM and single particle reconstruction. I will also induce defined functional states of the A. punctulata mGC and determine their structures to uncover the molecular mechanism of its activation and inactivation. Specifically, I plan to uncover how and in which stoichiometry the chemo-attractant resact is bound by the mGC complex; investigate which structural rearrangements are induced by ligand-binding in the extracellular domain; understand how extracellular and intracellular domains are structurally and functionally coupled; and unveil the structural and functional aspects of mGC modulation by phosphorylation and dephosphorylation. Phylogenetic comparison as well as conserved phosphorylation sites indicate that A. punctulata mGC is an ortholog of mammalian peptide-binding GC-A and GC-B. Therefore, these data will not only give us fundamental insights into the molecular mechanisms of chemo-attractant sensing in A. punctulata sperm cells, but also shed light on the molecular mechanism of mGC-based hormone signal transduction in eukaryotes.