Ligand modulation of sidechain dynamics in a wild-type human GPCR.
Ligand modulation of sidechain dynamics in a wild-type human GPCR.
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DOI：
10.7554/elife.28505
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发表时间：
2017-10-06
期刊：
影响因子：
7.7
通讯作者：
Rosenbaum DM
中科院分区：
文献类型：
--
作者：
Clark LD;Dikiy I;Chapman K;Rödström KE;Aramini J;LeVine MV;Khelashvili G;Rasmussen SG;Gardner KH;Rosenbaum DM
GPCR调节人体生理学的各个方面，生物物理学研究加深了我们对不同配体的GPCR构象调节的理解。然而，没有实验证据表明侧链动力学如何控制GPCR构象之间的变构转换。为了解决这一缺陷，我们产生了野生型GPCR（A2 AR）的样品，其除了在异亮氨酸δ1甲基处的1H/13 C NMR探针之外被氘代，这促进了用相对配体的1H/13 C甲基TROSY NMR测量。我们的数据表明，低[Na+]是必需的，以允许大激动剂诱导的A2 AR的结构变化，以及侧链动力学模式之间的激动剂（NECA）和反向激动剂（ZM 241385）结合受体，反向激动剂抑制快速ps-ns时间尺度运动在G蛋白结合位点显着不同。我们的GPCR NMR方法创建了一个框架，用于探索受体的不同区域如何通过调制快速ps-ns侧链动力学来响应不同的配体或信号蛋白。几乎人体的每一个方面--从我们的感官到我们的情绪--都以这样或那样的方式依赖于一个叫做G蛋白偶联受体的蛋白质大家族。这些受体蛋白，简称为GPCR，检测来自细胞外的信号并触发细胞内的活动。这使得细胞能够从周围环境中收集信息并相互交流。重要的是，由于GPCR调节体内参与疾病的许多过程，因此超过三分之一的批准药物靶向这些受体可能并不奇怪。像所有蛋白质一样，GPCR是长链样分子，具有重复的主链和称为侧链的短分支。每个侧链都有自己的化学性质和电荷，这可以影响链的不同部分如何相互作用以及蛋白质可以采用什么形状。这反过来又会影响药物或其他分子与受体蛋白结合的强度。蛋白质晶体学是一种用于更好地了解不同GPCR如何构建以及它们如何工作的技术。该技术涉及从纯蛋白质样品中生长晶体;这将数百万蛋白质拷贝锁定在适当位置，并提供其形状的快照。然而，GPCR-特别是它们的侧链-是灵活的，可以采用不同的形状，这不能通过只看蛋白质晶体完全看到。现在，Clark，Dikiy等人使用另一种称为核磁共振光谱的技术，或简称NMR，来了解药物如何影响GPCR中快速移动的侧链。首先，转基因酵母被用来创建一种称为腺苷受体A2 A的GPCR样品，这些样品被标记有特定的标记物，这使得通过NMR更容易测量蛋白质的结构和灵活性。这种方法揭示了样品溶液中过多的钠抑制了A2 A受体与药物结合时发生的大的结构变化。此外，它表明受体上几个区域的侧链以不同的方式移动，这取决于受体是否与激活药物或抑制药物结合。这些发现为理解侧链的运动如何帮助激活或抑制GPCR奠定了基础，并将补充正在进行的使用蛋白质晶体的研究。此外，生产标记蛋白质的新方法可以应用于其他类型的蛋白质，由于实际限制，到目前为止无法用NMR研究。在未来，这可能有助于科学家更好地了解药物如何影响这些蛋白质，并为一系列疾病开发新的治疗方法。
GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A2AR) that are deuterated apart from 1H/13C NMR probes at isoleucine δ1 methyl groups, which facilitated 1H/13C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na+] is required to allow large agonist-induced structural changes in A2AR, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics. Almost every aspect of the human body – from our senses to our moods – depends, in one way or another, on a large family of proteins called G-protein-coupled receptors. These receptor proteins, known as GPCRs for short, detect signals from outside the cell and trigger activity within the cell. This allows cells to gather information from their surroundings and to communicate with each other. Importantly, since GPCRs regulate many processes in the body that are involved in disease, it is perhaps unsurprising that over a third of all approved drugs target these receptors. Like all proteins, GPCRs are long chain-like molecules with a repetitive backbone and short branches called sidechains. Each sidechain has its own chemical properties and electrical charge, which can affect how different parts of the chain interact with each other and what shape the protein can adopt. This in turn can influence how strongly a drug or other molecule can bind to a receptor protein. Protein crystallography is one technique that has been used to better understand how the different GPCRs are built and how they work. The technique involves growing crystals from pure samples of the protein; this locks millions of copies of the protein in place and provides a snapshot of its shape. However, GPCRs – and especially their sidechains – are flexible and can adopt different shapes, which cannot be seen fully by only looking at protein crystals. Now, Clark, Dikiy et al. used another technique called nuclear magnetic resonance spectroscopy, or NMR for short, to understand how drugs affect the fast moving sidechains within a GPCR. First, genetically modified yeast was used to create samples of a GPCR called the adenosine receptor A2A that were labelled with specific markers which made it easier to measure the structure and flexibility of the protein by NMR. This approach revealed that too much sodium in the sample’s solution supresses the large structural changes that occur in the A2A receptor when it binds to a drug. Moreover, it showed that the sidechains of several regions on the receptor move in different ways depending on whether the receptor binds to an activating drug or an inhibiting drug. These findings lay the groundwork for understanding how the movements of sidechains help to activate or inhibit GPCRs, and will complement on-going studies using protein crystals. Moreover, the new approach to producing labelled proteins could be applied to other types of proteins that until now could not be studied with NMR due to practical limitations. In future, this may help scientists to better understand how drugs affect these proteins and to develop new treatments for a whole range of diseases.