Membrane signaling protein modulation by lipids and drugs

项目摘要/摘要 这个项目的目标是在原子水平上了解胆固醇如何调节完整的膜蛋白。 发信号。我们试图从两个生理上重要的生物学途径--胆固醇来解决这个问题 SREBP调节机制的动态平衡和G蛋白偶联受体的激素信号 (GPCRs)。SREBP机制以sCAP为中心，这是一种驻留在内质中的膜蛋白 网状结构。SCAP与SREBP转录因子结合并控制其在胆固醇依赖中的成熟 举止。目前还没有任何脊椎动物SCAP的原子结构，对详细的 关于SCAP的胆固醇结合或构象变化的生物物理水平。与SCAP一样，GPCRs也结合了配体 (例如，激素、神经递质和脂质)，并经历触发细胞内的构象变化 发信号。多个GPCRX-射线结构揭示了胆固醇结合的相互作用，然而功能 这些相互作用的意义在很大程度上是未知的。我们发现CB1大麻素受体可以结合 与其胆固醇结合部位重叠的膜包埋表面的变构调节剂。这 研究结果表明，胆固醇可能是CB1和其他GPCRs的变构调节剂。我们正在学习 胆固醇与SCAP和GPCRs的相互作用以更好地了解这种必需脂质是如何控制的 通过这些不同的膜蛋白发挥不同的功能。我们将使用低温电子显微镜来确定原子 全长脊椎动物SCAP的结构，要么与其共调控膜蛋白Insig结合，要么与可溶性的 SREBP2片段。这些结构将阐明这种脂质传感机器的体系结构，并提供 调节胆固醇的原子基础。同时，我们将获得第一批核磁共振波谱数据并冷却- 含和不含胆固醇的类天然磷脂纳米盘中GPCRs的EM结构数据。 这些数据将揭示嵌入的胆固醇是否可以稳定gpr的特定构象，以及 胆固醇相互作用是否维持在G蛋白信号复合体中。我们建议使用这一多样化的 一系列生物物理技术，以获得关于这些系统的胆固醇调节的前所未有的原子数据， 我们的发现可能被用来帮助开发通过 薄膜。

Project Summary/Abstract The goal of this project is to understand at the atomic level how cholesterol modulates integral membrane protein signaling. We seek to address this question in two physiologically important biological pathways, cholesterol homeostasis by the SREBP regulatory machinery and hormone signaling by G protein-coupled receptors (GPCRs). The SREBP machinery centers on Scap, a membrane protein that resides in the endoplasmic reticulum. Scap binds to the SREBP transcription factors and controls their maturation in a cholesterol-dependent manner. There are currently no atomic structures of any vertebrate Scap, and little is known at a detailed biophysical level about Scap's cholesterol binding or conformational changes. Like Scap, GPCRs bind ligands (e.g. hormones, neurotransmitters, and lipids) and undergo conformational changes that trigger intracellular signaling. Multiple GPCR X-ray structures have revealed cholesterol binding interactions, however the functional significance of these interactions is largely unknown. We found that the CB1 cannabinoid receptor can bind allosteric modulators at a membrane-embedded surface that overlaps with its cholesterol binding site. This finding suggests that cholesterol may be an allosteric modulator of CB1 as well as other GPCRs. We are studying the interactions of cholesterol with Scap and GPCRs to better understand how this essential lipid can control diverse functions through these different membrane proteins. We will use cryo-EM to determine the atomic structure of a full-length vertebrate Scap, either bound to its co-regulatory membrane protein Insig or a soluble fragment of SREBP2. These structures will elucidate the architecture of this lipid-sensing machine and provide an atomic basis for cholesterol regulation. In parallel, we will obtain the first NMR spectroscopy data and cryo- EM structural data for GPCRs embedded in native-like phospholipid nanodiscs, with and without cholesterol. These data will reveal whether embedded cholesterol can stabilize particular conformations of a GPCR, and whether cholesterol interactions are maintained in G protein signaling complexes. We propose to use this diverse array of biophysical techniques to obtain unprecedented atomic data on cholesterol regulation of these systems, and our findings may be used to help develop drugs that modulate Scap or different GPCRs through the membrane.