Trends in pharmacological science, Volume 36, Issue 1, p22–31, January 2015 Abstract Generic residue numbers facilitate comparisons of, for example, mutational effects, ligand interactions, and structural motifs. The numbering scheme by Ballesteros and Weinstein for residues within the class A GPCRs (G protein-coupled receptors) has more than 1100 citations, and the recent crystal structures for classes B, C, and F now call for a community consensus in residue number- ing within and across these classes. Furthermore, the structural era has uncovered helix bulges and constric- tions that offset the generic residue numbers. The use of generic residue numbers depends on convenient access by pharmacologists, chemists, and structural biologists. We review the generic residue numbering schemes for each GPCR class, as well as a complementary structure- based scheme, and provide illustrative examples and GPCR database (GPCRDB) web tools to number any receptor sequence or structure. Alignment of the class-specific generic residue numbers based on structural alignment of crystal structures of representative receptors from class A (bovine rhodopsin, bRho), B (glucagon receptor), C (metabotropic glutamate receptor 1, mGlu1), and F (Smoothene, SMO). Class-specific reference residue positions (X.50) for each of the seven TM helices are given in bold font. Full text
G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway Xin Zhou, Zhen Wang, Wei Huang and Qun-Ying Lei Acta Biochim Biophys Sin 2015, 47: 10–15; doi: 10.1093/abbs/gmu108 Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers. 图例: GPCR对Hippo-YAP/TAZ通路的调节作用 全文: http://abbs.oxfordjournals.org/content/47/1/10.full Go to ABBS Volume 47, Number 1, 2015 : a special issue on Hippo signaling
Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway. Shuguang Yuan, Slawomir Filipek, Krzysztof Palczewski, Horst Vogel Nature Communication doi:10.1038/ncomms5733 full text news story A schematic view of three different rotamer conformations of the highly conserved Y7.53 residue. (a) The YII conformation is preferred in activated GPCRs; during formation it is accessed by extracellular water molecules forming a continuous internal water network. (b) For receptors similar to Rho and S1PR1 the YII state is associated with the cytoplasmic influx of water. GPCRs are shown in grey and the G-protein subunits are displayed in different colours. (c) The YI conformation is preferred in inactive GPCRs with two hydrophobic layers blocking a continuous water pathway. (d) The YIII conformation is preferred in GPCR meta states with one hydrophobic layer located next to the NPxxY motif and blocking formation of a continuous water pathway. Abstract Recent crystal structures of G-protein-coupled receptors (GPCRs) have revealed ordered internal water molecules, raising questions about the functional role of those waters for receptor activation that could not be answered by the static structures. Here, we used molecular dynamics simulations to monitor—at atomic and high temporal resolution—conformational changes of central importance for the activation of three prototypical GPCRs with known crystal structures: the adenosine A 2A receptor , the β 2 -adrenergic receptor and rhodopsin . Our simulations reveal that a hydrophobic layer of amino acid residues next to the characteristic NPxxY motif forms a gate that opens to form a continuous water channel only upon receptor activation. The highly conserved tyrosine residue Y 7.53 undergoes transitions between three distinct conformations representative of inactive, G-protein activated and GPCR metastates. Additional analysis of the available GPCR crystal structures reveals general principles governing the functional roles of internal waters in GPCRs.
Published Online September 12 2013 Science Express Science DOI: 10.1126/science.1241475 Report Structure of the CCR5 Chemokine Receptor–HIV Entry Inhibitor Maraviroc Complex full text Abstract The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here, we report the 2.7 Å resolution crystal structure of human CCR5 bound to the marketed HIV drug Maraviroc. The structure reveals a ligand binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor/gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.
1. Crystal structure of pre-activated arrestin p44 Yong Ju Kim,Klaus Peter Hofmann,Oliver P. Ernst,Patrick Scheerer,Hui-Woog Choe Martha E. Somme nature 497,142–146 (02 May 2013)mdoi:10.1038/nature12133 Abstract Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins 1 , 2 and initiate G-protein-independent signalling 3 . Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs 4 . Structures of the inactive state of arrestin are available 5 , 6 , but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a ~21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor binding loops in the central crest region include the finger loop 7 , 8 , 9 , loop 139 (refs 8 , 10 , 11 ) and the sequence Asp 296 Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system. full text 2. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide Arun K. Shukla,Aashish Manglik,Andrew C. Kruse,Kunhong Xiao,Rosana I. Reis,Wei-Chou Tseng,Dean P. Staus,Daniel Hilger,Serdar Uysal,Li-Yin Huang,Marcin Paduch,Prachi Tripathi-Shukla,Akiko Koide,Shohei Koide,William I. Weis,Anthony A. Kossiakoff,Brian K. Kobilka Robert J. Lefkowitz Nature497,137–141(02 May 2013)doi:10.1038/nature12120 Abstract he functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins 1 . G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors 2 , and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization 3 . Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways 4 . Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5 ). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1–V2Rpp–Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the ‘lariat loop’ implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins. full text
Structure of the human glucagon class B G-protein-coupled receptor Fai Yiu Siu,Min He,Chris de Graaf, et.al. Nature (2013) doi:10.1038/nature12393 Abstract : Binding of the glucagon peptide to the glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting; thus GCGR plays an important role in glucose homeostasis. Here we report the crystal structure of the seven transmembrane helical domain of human GCGR at 3.4 Å resolution, complemented by extensive site-specific mutagenesis, and a hybrid model of glucagon bound to GCGR to understand the molecular recognition of the receptor for its native ligand. Beyond the shared seven transmembrane fold, the GCGR transmembrane domain deviates from class A G-protein-coupled receptors with a large ligand-binding pocket and the first transmembrane helix having a ‘stalk’ region that extends three alpha-helical turns above the plane of the membrane. The stalk positions the extracellular domain (~12 kilodaltons) relative to the membrane to form the glucagon-binding site that captures the peptide and facilitates the insertion of glucagon’s amino terminus into the seven transmembrane domain. PDB: 4L6R full text: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12393.html
The structure of the chemokine receptor CXCR1 in phospholipid bilayers was released these days in Nature Letters. This is the first NMR GPCR structure and The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiologicalconditions. Such features may provide a more close view towards GPCR's function. Structure of the chemokine receptor CXCR1 in phospholipid bilayers Sang Ho Park,Bibhuti B. Das,Fabio Casagrande,Ye Tian,Henry J. Nothnagel,Mignon Chu,Hans Kiefer,Klaus Maier,Anna A. De Angelis,Francesca M. Marassi Stanley J. Opella Abstract CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth 1 , 2 , 3 . IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation 2 . CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors 4 , 5 , 6 , 7 . Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences 8 , as well as addition of stabilizing antibodies and small molecules 9 that facilitate crystallization in cubic phase monoolein mixtures 10 . The intracellular loops of GPCRs are crucial for G-protein interactions 11 , and activation of CXCR1 involves both amino-terminal residues and extracellular loops 2 , 12 , 13 . Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity 14 . Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer. full text link http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11580.html
今天半夜两点钟(美国西部时间)的时候，我被香港大学一个同学急促的电话吵醒，他激动地对我说，你的老板获得诺贝尔奖了！我当时非常地惊讶，虽然很多人预测Brian Kobilka会获得诺贝尔奖，但是我真得没有想到会这么早。估计我是实验室里除了Brian和他的夫人Tongsun之外知道这个消息最早的人了。最近一年来我一直学习导师Brian的做事风格踏实专心地做科研，虽然课题非常challenging（挑战性），但是潜移默化地受到Brian的感染，对于自己的课题始终抱有信心，当然希望最终会取得成功。记得施一公教授曾在苏州冷泉港会议给我们说，Brian是一个真正的hero（英雄）。随着自己研究和与Brian的交流的日益深入，我越来越体会到这个词的分量。于是我按捺不住想说几点我的感想，纯粹杂谈，没有什么特别的逻辑性。更希望我们这些资历尚浅的后生能从他身上学到科学研究所应该具备的一些可贵品质。 一、HHMI也有看走眼的时候 GPCR（G蛋白偶联受体，G-protein coupled receptors）作为人类基因组编码的最大类别膜蛋白超家族，有800多个家族成员，与人体生理代谢几乎各个方面都密切关联【1】。它们的构象高度灵活，调控非常复杂，天然丰度很低，起初非常难以研究。美国杜克大学的Robert J. Lefkowitz(与Brian分享诺奖并是他的博士后导师)在这个领域做了非常多开创性的工作和贡献。Brian就是在Robert的实验室里首先克隆了人Beta2肾上腺素受体的cDNA序列，开始了与GPCR研究的不解之缘【2】。自从Brian在斯坦福大学医学院开始独立研究以来，十几年的时间里一直没有重大的突破，导致HHMI（霍华德休斯医学研究所，Howard Hughes Medical Institute）这种擅长支持长期高风险研究的机构都没有耐心了。HHMI于是在2003年左右的时候停掉了Brian的funding。那几年确实非常艰苦，并且由于课题风险太高，Brian都不敢也没有条件招收postdoc。不得已他只能自己做实验，不过幸亏那几年Brian还是靠着NIH（美国国立卫生研究院，National Institutes of Health）的R01资助坚持了下来，并于2007年解析了第一个人非视紫红质GPCR的晶体结构，取得了初步的成功【3】。当我们看到他现在的光环时，很少有人能体会到他过去的艰辛。现在回首看来，我认为一方面是默默地坚持，拥有一颗强大的心脏承受失败，对于科研非常重要；另一方面，我们也看到，HHMI也有看走眼的时候~我觉得美国有时候很奇怪，我知道的有时即使是诺贝尔奖获得者，也不能保证funding（科研资助）每年都能顺利renew（更新）。虽然Brian现在每年都还在担心funding的问题（估计是被那几年搞怕了），但是我相信按照现在实验室的研究势头在今后的几年应该不会是个问题吧。 二、Brian Kobilka获得诺奖的主体工作 我听到不少人说，GPCR结构生物学领域最终肯定会获得诺奖。这里不得不佩服饶老师看待当前生命科学前沿动态的精准眼光【4】。在GPCR这个竞争日趋激烈的领域，Brian确实是一个几乎完美的成功者。举个例子，当人遇到危险或紧张时，肾上腺会分泌一种激素，叫肾上腺素。它作为配体，犹如一把钥匙，高度特异性地打开或者激活 肾上腺素受体 这把锁。然后细胞就能感受到这一外部的危险，并快速做出相应的生理反应。比如 心跳变快， 瞳孔放大，呼吸加速，肌肉收缩等来应对，即所谓的fight-or-flight（迎战或逃跑）。 Beta2肾上腺素受体一直是研究GPCR最标准的模式受体。Brian就是围绕这个受体开展了长达二十多年的工作。厚积薄发，在最近的几年里才有最为出色的突破和进展。这些成果为GPCR领域很多一般性的重要生物学问题提供了重大的启示，并同时为基于GPCR结构的药物开发起着重要的推动作用，因此成为很多国际大型制药公司争相追逐的热点。虽然Brian在其它GPCR领域也做出了很多出色成果，但是围绕 Beta2肾上腺素受体相对是最完备的工作，我猜想这应该是今年Brian接受诺贝尔奖的主体工作吧。简单列举如下： 1）Brian在2007年与Raymond研究组合作解析了非活化 Beta2肾上腺素受体的晶体结构。Brian在这个成果上的主要贡献是基于首次发明了T4溶菌酶融合的技术对该膜蛋白做了长期大量的纯化制备和生化研究，Raymond研究组则主要负责数据收集和结构解析工作。另外，Brian还在那年独立地利用抗体稳定技术解析了这个膜蛋白。这让人们看到了神秘多年的 第一个非视紫红质GPCR的全貌； 2）Brian在2011年初解析了第一个真正处在活化状态的激动剂（agonist）结合GPCR的晶体结构（活化状态的GPCR非常活跃，更难以结晶）【6】。这里我用‘真正’一词是因为这个结构是借助于Nanobody抗体技术筛选出来的、构象选择性的、功能得到充分鉴定的G蛋白类似物帮助结晶，让人们第一次可以通过和非活化状态结构的比较来了解GPCR激活的结构生物学机制； 3）Brian 在2011年中 解析了第一个激活状态下GPCR和下游G蛋白复合物相互作用瞬间的晶体结构【5】（这个成果是在施一公教授组织的2011年苏州冷泉港会议上关于膜蛋白主题报道的，大部分顶尖的膜蛋白相关领域科学家都出席，当时震惊全场）。它让人们第一次观察到细胞外的小分子配体如何通过灵活多变的GPCR进而激活胞内体积相对巨大的G蛋白，从而传递各种各样的生理学信号。这个结构重要的科学价值我相信通过今后的引用率会慢慢得到证明，应该也是这次诺贝尔奖的皇冠之作。估计外行的人看到都会觉得很美； 4）以方法学而论，Brian为了解析GPCR及其与G蛋白的复合物结构，他和合作者一起开发和应用了若干创新的技术手段，如抗体稳定（nanobody），T4溶菌酶融合（T4 lysozyme fusion）、超强激动剂（super agonist）、共价偶联配体（covalent ligand）、新型去垢剂（new detergent type）、单分子三维电镜分析（single particle EM analysis）、改进的适用于GPCR的脂立方相晶体筛选、微束衍射（micro-diffraction）等下游很多晶体学tricks（技巧）。我相信这些技术以后也会让相关的膜蛋白结构生物学研究受益。 我刚加入实验室的时候，曾经也想向Brian提出做GPCR里一个新的A类家族的结构。按照通俗的话来说，这样做性价比很高，方法和套路在实验室非常成熟，如果运气好，可以进展非常快，有可能半年以内的时间就拿到一个inactive（非活化）的GPCR结构。按照现在的情况，肯定还是CNS（Cell/Nature/Science）级的论文。但是Brian婉拒了我的想法。他现在的研究策略是专注于三类代表性的GPCR家族，深入去做。以一些模式受体来回答这个领域里尚未回答的一些重要生物学问题。 三、承载科学家使命的Dr. Kobilka 这里Dr.我想指的更多是医生的意思。很多人知道，Brian Kobilka毕业于耶鲁大学医学院，拿到的是MD医学博士学位。因为在美国拿到MD学位的人大部分都去做了医生，因为医生在美国是一个有地位、受人尊敬并且收入高的职业。Brian也在华盛顿大学做了几年内科住院医生，但是基于对科学的热爱，毅然决然地踏上了充满各种未知的科研之路。大家可以想见的是，Brian当然没有在学生时期上过结构生物学的课程。但是他为什么能够在结构生物学的领域如此成功呢？我觉得这主要归结于他对待科学的态度、多领域的研究背景和科学的思维方式。很多人应该不知道，Brian在自己实验室早期从事了大量的细胞生物学、生理学和生化的研究工作。他在基于转基因小鼠的心脏生理研究上其实也做出了很出色的工作。加上好奇心驱动，他对相关的广泛领域都有所涉猎，具有广泛的知识面和对多个领域技术研究特点的出色理解。这让他在近几年来投身的结构生物学领域具备了扎实的基础。 有句古话，知己知彼，百战不殆。正是他这么多年积累的对于GPCR的各种性质特点充分的理解，他对哪些因素有可能阻碍GPCR晶体的生长和结构的解析有着敏锐的洞察力，而不是只依赖于晶体学本身技术和原理的认识。比如，大家知道，膜蛋白大部分是疏水的部分，这些地方是非常不利于每个蛋白质分子排列的。就像无法相互契合的砖头很难盖得好一栋完整的房子。那么自然地就是能够想办法增加膜蛋白表面的极性面积，让分子彼此之间倾向于相互靠拢，那么这栋房子就更容易盖好了。再比如，GPCR分子是高度活跃的，就像一个个很调皮活泼的孩子，让他们一个个乖乖地服从命令站好队不是那么容易，那么就要想办法能够让他们安静下来。比如给每个孩子一个好吃的糖果，让他们乖乖地听话，这样就更容易让他们服从安排。那么特定的配体就像这个糖果，让GPCR分子安静下来，不那么活跃。总之，正是基于对于GPCR分子特性的很好理解，Brian能够想出很多为GPCR晶体的生长和改善的ideas（主意）。这让我同时也想到好几年前一篇有名的科学博文“从纯化到星辰”里作者在冷泉港实验室进修的体会：真正的科学家应该不会因为过去受到的科研训练所束缚，应该随时准备好根据研究的需要学习新的东西。我觉得Brian在这点上同样是我们学习的榜样。 四、谦逊、害羞并可爱着的Brian 人常说，性格决定命运。我一直很相信这句话。虽然人无完人，但在我看来，Brian确实是接近于一个完美的人。这是我发自内心的看法。他做事低调，性格温和，为人谦虚，坚韧执着。同时，我们也看到他是一个非常合格的导师，丈夫和父亲。要真说缺点，他不太擅长和别人谈论科研以外的话题，对媒体的访问也不适应，甚至于紧张。在实验室这么久来，从来没有看到他因为任何事情对学生语气生硬过，更别说生气。他总是非常顾忌其他人的感受，如果实验室有人粗心犯了什么错误，他不会先选择直接去找这个学生说，而是会在组会上作为一个一般性的问题提出来。他的性格内敛到以至于学生有时候都会觉得不好意思。比如有时候他很想知道我一段时间的进展，他会静静地站在我的面前，看着我不好意思地微笑，欲言又止，我明白他是想让我tell something给他。我从他身上没感觉到一丁点有些大牛老板的那种push（施压）。在我看来Brian是一个非常pure（纯粹）的scientist（科学家）。 五、兴趣和工作的区别 我也常听到科研界的同事说，做科学的境界主要分为两种，一种是真心地喜欢，作为自己热爱的事业去做，是兴趣驱动地；另外一种是作为谋生的一种手段，只是选择的一个job（工作）而已。很明显从事科研的人的理想是前一种。但是往往理想很丰满，现实很骨感。由于各种主观和客观的因素，大部分都是处于后者的阶段。有时候追逐理想但远离现实时会付出很多代价，放在科研上也是如此。比如有时焦头烂额地应付各种资金申请和评审可能会把对于某个领域的热爱抛在脑后。我能一直感受到Brian对科学那种inherent（内在）的热爱。他一直被好奇心驱使着想要回答一些重要的生物学问题。他和我讨论课题时，经常使用的一个句式是：‘I am very interested in …’来表达他希望我关注并且回答的问题。实验室的每个同事都被Brian对科研的这种热爱所感染，都在享受着艰辛科研工作中所带来的乐趣。 六、Brian和他的合作者们 细心的听众可能会注意到，每当Brian在媒体面前发表所谓获奖感言时，他一直都特别强调众多合作者的贡献【8】。他说对于他工作的认可，也相当于对于合作者工作的认可。如果能了解一下Beta2肾上腺素受体和G蛋白的复合物结构，大家应该能认识到，没有很多合作实验室强大的协助，单靠一个实验室，要解析这个结构是非常困难，甚至在短期内是不可能的。坦诚地说，Brian的GPCR研究涉及到非常多前沿的技术，而往往一个实验室很难在多方面都非常专业。所以需要多方面出色的合作者一起共同来解决一些challenging的问题。但是同时会出现另外一个问题，为什么这么多出色的科学家愿意与Brian合作？根据我的了解，Brian遇到非自己专长的知识范畴时，他会如同一个学生一样，非常谦虚的主动联系相关的研究者。即使之前完全不认识，也不管对方的资历是深或是浅。他一直非常肯定合作者的贡献，并且给予合作者公平的承认，让每个合作者都心悦诚服，有时候那种不耻下问的精神非常值得学习。 七、革命尚未成功，同志仍需努力 有不少人评价Brian的工作完整地诠释了单个GPCR非活化、活化及其G蛋白复合物的结构生物学基础，系统地阐述了配体调控的G蛋白激活机制。虽然这些都是里程碑式的工作，对于GPCR领域会有巨大的推动作用。并且可以预见，GPCR作为近年来的明星分子，肯定会继续保持热度并让许多研究者投身于这个领域。但是还是有非常多基础性的重要问题还没有得到解决，比如G蛋白选择性激活的机制、非G蛋白调控通路机制(beta-arrestin)和GPCR dynamics特性的系统分析等等。正如我上面所说，Brian对于以后的研究方向有着清晰的规划，将针对一些特定的生物学问题，选择相应代表性的对象进行研究。我们坚信接下来的工作将会对这复杂而美妙的GPCR调控全景进一步进行完善。 后注1：特别需要强调的是获得诺奖的工作主要是已经离开实验室的两个博士后Soren和Daniel完成的，他们是学习的榜样，谨在此向这些师兄们致以敬意; 后注2：有个别朋友指出我这篇文章里混杂少量英文单词，虽然我也不喜欢中英文混杂的方式，因为在我看来中文语言博大精深，没有什么用中文难以表达的意思。只不过我觉得这些英文单词稍微能更精确和忠实地体现我想表达的意思。比如‘challenging’是科研领域里经常形容课题难度和风险高、‘hero’是施一公教授评价Brian的原词、‘funding'和'renew'是美国申请科研经费经常用到的术语、'push'也是我们中国学生经常形容一个老板喜欢对自己的工作施加压力等等。我都在后面注明相应意思，我觉得大部分同学应该是会认同我的观点吧。 参考文献： 【1】Fredriksson R, Lagerström MC, Lundin LG and Schiöth HB. The G-Protein-Coupled Receptors in the Human Genome Form Five Main Families. Phylogenetic Analysis, Paralogon Groups, and Fingerprints. Mol Pharmacol. 2003 Jun;63(6):1256-72. 【2】Kobilka BK, Dixon RA, Frielle T, Dohlman HG, Bolanowski MA, Sigal IS， Yang-Feng TL, Francke U, Caron MG, and Lefkowitz RJ. cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor.Proc Natl Acad Sci U S A. 1987 January;84(1): 46–50. 【3】Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science. 2007 Nov 23;318(5854):1266-73 【4】http://blog.sciencenet.cn/home.php?mod=spaceuid=2237do=blogid=621167 【5】Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature. 2011 Jul 19;477(7366):549-55 【6】Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK. Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. Nature. 2011 Jan 13;469(7329):175-80. 【7】http://gpcr.scripps.edu/ 【8】Buchen L. Cell signalling: It's all about the structure. Nature 476, 387-390 (2011) 斯坦福大学诺奖新闻发布会现场，右边坐的是斯坦福大学校长，左边坐的是医学院院长。 不久前在接受一个科研慈善基金会庆祝仪式上的实验室全家福（老板左右两侧笑得最灿烂的是实验室仅有的两个工作极其勤奋并非常有科研天赋的PhD学生。他们的出现让我彻底改观对于美国学生不如中国学生勤奋努力的看法） 获奖后Brian和太太在空间狭小的办公室门前合影，屋里只有桌子和电脑。相邻的小隔间是实验室的生活区和Lab manager的办公地方。
有严重知识错误的精选博文“ G 蛋白研究再获诺贝尔奖” 1994 年生理或医学奖奖励 G 蛋白及其工作原理的工作 2012 年化学奖奖励 G 蛋白偶联受体的结构解析的工作。 而博文中将“ G 蛋白”、“ G 蛋白偶联受体”、“ G 蛋白受体”混为一谈。 根本不存在“ G 蛋白受体”这样的说法，“ G 蛋白”不是“ G 蛋白偶联受体”的配体。“ G 蛋白”与“ G 蛋白偶联受体”是完全不同的蛋白家族，它们之间的关系如下图所示（图片来自网络）：
The Nobel Prize in Chemistry 2012 was awarded jointly to Robert J. Lefkowitz and Brian K. Kobilka "for studies of G-protein-coupled receptors (GPCR) http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/ Robert J. Lefkowitz Born: 1943, New York, NY, USA Affiliation at the time of the award: Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA Prize motivation: "for studies of G-protein-coupled receptors" Brian K. Kobilka Born: 1955, Little Falls, MN, USA Affiliation at the time of the award: Stanford University School of Medicine, Stanford, CA, USA Prize motivation: "for studies of G-protein-coupled receptors"
一个可能的诺贝尔化学奖 http://blog.sciencenet.cn/blog-2237-433929.html 我们知道：诺贝尔化学奖委员会，不时地肯定化学和生物交叉的工作，比较常见的是生物化学和生物物理学的工作，有时也给分子生物学。 从2003到2009之间7年的诺贝尔化学奖，有5年给生物学研究：2003年钾通道的结构和水通道，2004年蛋白质降解，2006年基因转录的结构生物学研究，2008年绿色荧光蛋白，2009年合成蛋白质的核糖体结构。 结构生物学占了很大比重（2003、2006、2009）。 我们也知道：诺贝尔化学奖委员会经常犯错误，不该给的他们给了、该给的他们没给，两种错误都犯过。 2003年，不应该奖水通道的发现，因为并不足够突出：不是第一个通道（是第几十个通道）、也无特殊性。 2006年，化学家们只重视自己懂的，而忽略了同一科学领域中偏生物、但更重要的工作。基因转录领域，有两项工作的重要性毫无疑问高于解出转录因子的X线 晶体结构：发现第一个转录因子（Mark Ptashne）、发现RNA多聚酶（Robert Roeder）。但诺贝尔化学奖委员会过分强调结构而忽略了转录领域中更重要的生物学工作。 基本可以放心：化学奖委员会一如既往地跨界出现错误，既不是第一次，也不会是最后一次。 不过，化学奖委员会继续给结构生物学发奖时，如果做到一个中等偏上的研究生的水平（比如本文就是给研究生上课过程中两句带过，也是中上研究生可以写出来），就可以公平地奖励一个大家都会公认的工作。谈不上将功补过，可以证明他们不都经常肤浅。 可以奖对于GPCR（G蛋白偶联受体）的结构生物学研究。 GPCR是细胞膜的跨膜蛋白，一般来说，把细胞外的信号转入细胞内。 GPCR的发现历史很长。第一个是在19世纪发现于眼睛视网膜上。1851年，Heinrich Müller发现视网膜红紫色，认为是血红蛋白造成。年轻的德国医生Franz Boll(1849–1879)实验证明视网膜漂白并提出其物质基础是“红紫物质”，存在于视杆细胞，进行光化学反应。不幸他因肺结核而英年早逝。 Boll发表1877年论文不久，德国医生Willy Kühne很快继续其研究，大量投入时间和精力，在1878到1882年间发表22篇论文，将红紫物质称为“视紫”（visual purple），发现光化学还原，并用胆盐提取了视紫，也就是后来大家所谓的“视杆蛋白”(rhodopsin)。Kühne提出，光解构视杆蛋白，解构 的光化学反应产物刺激视神经。 以后实验证明，视杆蛋白确实对视觉非常重要。 从生物化学和生物物理学角度来说，这是第一个细胞膜蛋白。不仅对于理解视觉有推动，而且有助于以后研究和理解其他一些膜蛋白。很长时间，这是唯一被较多人研究的膜蛋白。 视杆蛋白不仅存在于有视觉的高等动物，也存在于细菌中：用于感光，虽然不能形成视觉。 哺乳类的视杆蛋白由约350个氨基酸连接组成。到1970年，洛杉矶加州大学的研究者获得其9个氨基酸顺序，1977年美国的Hargrave获得其16 个氨基酸顺序。1983年，通过分子生物学帮助，Hargrave等和俄国的Ovchinnikov等分别推出牛视杆蛋白的全顺序。 1960到1980年代，发现G蛋白调节很多递质和激素的受体，这些受体就都称为GPCR（G蛋白调节受体），氨基酸顺序类似于视杆蛋白。因为发现G调节 蛋白和提出GPCR概念，美国的Alfred Gilman和Martin Rodbell获1994年诺贝尔生理或医学奖。 这样，研究视杆蛋白和研究一般GPCR实质是同一类研究，差别只在于视杆蛋白参与细胞对光的反应、其他GPCR一般来说参与细胞对细胞外化学分子的反应，2011年3月发现果蝇视杆蛋白可能参与对温度反应。 用X线晶体衍射研究蛋白质的空间三维结构，是理解蛋白质功能的一个重要途径，可以在分子和原子水平上理解生物分子如何起作用，还可以通过结构提出合理的方 法设计新的药物，所以一直是生物与化学/物理交叉的一个重要领域。不仅以上提到的2003年以后多次诺贝尔化学奖给结构生物学，以前也较多，如：1962 年Max Perutz和John Kendrew，1964年Dorothy Hodgkin，1982年Aaron Klug，1988年Johann Deisenhofer，Robert Huber和Hartmut Michel，1997年 John Walker等，当然，这些奖也并非个个没有争议，但1962和1964的是大家公认的重要工作。 对于视杆蛋白/GPCR的结构生物学研究，几乎肯定会获得诺贝尔奖。 1997年，日本Kimura等解出了细菌的视杆蛋白结构。2000年美国的Palczewski等解出牛视杆蛋白的结构。2007年美国斯坦福大学的 Brian Kobilka和Scripps研究所的Raymond Stevens解出也是GPCR类的b肾上腺素受体的结构。其后他们和一些实验室不断解出新的GPCR结构，以及GPCR结合激动剂、抑制剂以后的结构。 目前，解GPCR的文章在Nature、Science上如雨后春笋。 从工作重要性来说，早期的里程碑非常清晰，后面的不是每次都是一个人的工作，但相对来说可以看到有些贡献比较突出，如：获得视杆蛋白氨基酸序列贡献最大 是美国的Hargrave，最初解细菌视杆蛋白结构是日本大阪生物分子工程研究所的Yoshiaki Kimura，第一个解动物视杆蛋白结构的Palczewski，第一个解非视杆蛋白的GPCR结构的Brian Kolbika。 实际上，如果诺贝尔化学奖委员会水平稍微提高一点，1997年解细菌视杆蛋白的Yoshiaki Kimura应该于2003年获奖。那年，因为1998年第一个解钾通道蛋白的MacKinnon获奖。2003年的奖应该给MacKinnon和 Kimura，而不应该给做水通道的工作，因为MacKinnon和Kimura分别解出两个非常重要的膜蛋白结构。当然，化学奖委员会水平有限，只知道 跟踪生物的热点，钾通道解完后，立即受到大家重视，化学奖委员会就知道重视，而视杆蛋白那时没有热起来，所以化学奖委员会就不知道自己做功课了。 最近几年，因为GPCR受体结构非常热门，所以，水平如化学奖委员会也会知道，所以肯定会给。 不过，纵观其历史失误率，也可以猜想它还很可能犯错误，比如忽略生物学重要的Hargrave做的一步，或再度忽略Kimura的工作，而只给做牛视杆蛋白和后面GPCR的科学家、甚至只给做GPCR受体的。 无论这个委员会怎么犯错误，对于稍花点时间看这个领域的人来说，发现重要的工作并非难事。 在视杆蛋白是冷门的时候，没有几个实验室竞争研究其结构。在GPCR没有解出一个的时候，竞争也不多。现在成为热点，解一个GPCR结构发一篇文章、吸引一批读者和引用的时候，真正突破的，还是以前几个主要工作。 从生物学机理理解需要来看，结构生物学将在可以预见的将来继续发挥很大作用，其中经典的X线衍射结构分析，也会继续很有用。如果今后能够做大分子活体 结构、动态结构、在生物体系中观察结构变化，而不局限于结晶的分子、水中的小分子，那么广义的结构生物学将起更大作用。 Müller R (1851). 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As the most important membrane protein, GPCR MD simulation is a quite hot topic nowadays. However, how to assemble protein/membrane system and which forcefield to assign for the whole system are two main critical and tough task for GPCR simulations. 1. Assemble protein/membrane system Many tools are now available for protein/membrane building including: VMD (http://www.ks.uiuc.edu/Research/vmd/) CHARMM-GUI (http://www.charmm-gui.org/) Desmond System Builder (http://www.deshawresearch.com/resources_desmond.html) g_membed (http://wwwuser.gwdg.de/~ggroenh/membed.html) InflateGro (http://moose.bio.ucalgary.ca/index.php?page=Translate_lipdis) VMD can support well for POPC and POPE membrane system building with CHARMM27 and CHARMM36 FF. H owever, one have to add additional solvent and i ons into the syst em by tcl script from VMD tutorial. It will also n eed some script to merge membrane and protein. Moreover, since the lipids are not pre-equilirated, it would be necessary to equ ilibrate the whole system at least 20 ns before MD production. CHARMM-GUI aims to provide more convenient way for NAMD or CHARMM sim ulation. It can gene rate a embeded protein/memb rane system with OPM position through web page interface. It even can helps to assi gn CHARMM CGFF for the ligand. H owever, the re are also some o bvious week nees for it: there are so many atom clashed between lipids that we can hardly believe the lipids are pre-eq uilib rated which claimed by the author; the input file provided by CHARMM-GUI is not good enough for membrane protein simulation s ince the whole quilibration step on ly contains no m ore than 3 ns and obvious GPCR helix movement are of ten observed w ithin such short time which shouldn't expected at this time scale level. So, one have to improve the protocol by himself. Desmond System builder tool is incorporated in Schrodinger Maestro GUI and it provides very friendly interface for users. It can build a OPM based position for pr otein/membrane system very easily by c licking some bottoms . It can also assign CHARMM36 FF by VIPARR tool in D esmond. Both g_membed and InflateGro are tools w ithin Groma cs and both of them can embed the prot ein into a pre-equlibrated membrane system which save lot of ti me for equilibration . Although g_membed is a little bi t diff icult than InflateGro, but the output seems to be much better than the later one. 2. Force filed It is said that CHARMM36 FF is the best FF for lipids which i s currently the only FF can reproduce lipid gel phase property. Howe ver, recen t Lipid 11 FF from latest Amber 12 is also claimed to be as good as CHARMM36 FF , al though related p aper is being revi ewed th ese days. Both full atom FF are quite good option for protein system simulation. There are also other FF and me thods including Gromos FF which is a united atoms FF and nowadays coarse gain MD which use dum my sphere to represent groups and acce lerate the simulation dra matically (24 core workstation can even achieve up to several microsecond/day ). The demerit for those methods are also obvious: we gain w hat we paid. 3. Top ology for Ligand T his is always a head ache problem for many people in MD simulation. In Desmond, ligand to pology can be recognized automatically with OPLS_2005 FF. H owe ver, OPLS_2005 is only go od e nough for t ens of ns MD simulation, it is rather poor if su b mit to micro se cond MD. If we would like to use CHARMM36 FF for protein/membrane system bound with ligand, we can generate to pol ogy from S wissparam (http://swissparam.ch/) and convert them into Desmond Viparr format by script from Desmond 2012 ($SCHRO DINGER/desmond-v31023/data/viparr/converters ). This tool sometimes doesn't work well, it is said that it can be full y supported by D esmond in the next version of Desmond which would be released at the beginning of next year . What's n eed to mention is that CHARMM C G FF is also reachable in Des mond 2012 ($SCHRODING ER/desmond-v31023/data/viparr/ff/cgenff_base_v2b7 ), one can build manually with those molecul ar templates if the target on e is not so complicated. CHARMM CG FF also could be obtained from (https://www.paramchem.org/). If the ligand structure is not so complicated, it may work well. H owever, it some time s may not re cognize ligand bond order and so on correctly. In this way, one have to go to CHARMM forum for helps. Amber GAFF is definitely ext re mely at tractive and the primary choice for a ligand bound system. When the latest LIPID 11 FF in Amber 12 come out, Ambe r should be the first choic e for many people especially those work with ligand. 4. Efficiency Alt hough the hardware of compu ter deve lop s so fast nowada ys that CPU update one generation almost each year, the e ffici ency of MD simulations seems don't imp rove so much these days. F or instan ce: no matter how many CPU we use , for a typical mem brane protein simulation (13 2 lipids, 300aa protein, 50,000 at oms in all ) with full atom FF and typical cutoff (9-10) with PME: Gromacs can get up to 2 0 ns/day (double precision), Amber 12 ns/day, NAMD 4ns/day. Desmond is an exception since the parralization is much more superior tha n any other MD tools, it can up to 100 ns/day with 512 CPU. I t can even up to several microseco nd /day in A nton with full atom FF. It woul d be a wise option to use either D esmond or Gromacs , if one would like to run hundre ds of ns with full atom FF. Of course, it is also accepta ble for Amber and NAMD CPU performance if the simulation on ly last for tens of ns. GPU technology is developing quite fast in recent one or two years and it also bring exc iting news for computational work especially NVIDIA CUDA accel erations. F or instance, with two GTX590, A mber 12 can get up to 20ns/day while 24 core i7 3.6 GH z CPU can only get 4ns/day ( with intel com piler , gnu is even m uch slower). NAMD on the other hand, can get 5ns/day with CUDA acc eleration w hile 24 core i7 3.6 GHz CPU can only get 0.5 ns/day. C urrently, GPU calculation is not supported in Desmond, but this feature is e x pected to be available in next version.
标签: GPCR
