International Collaboration Finds 3D Structure of Key Drug Target for Diabetes
LA JOLLA, CA – July 17, 2013 - An international team led by scientists at The Scripps Research Institute (TSRI) has determined and analyzed the three-dimensional atomic structure of the human glucagon receptor. The receptor, found mainly on liver and kidney cells, helps regulate glucose levels in the bloodstream and is the target of potential therapeutic agents for type 2 diabetes.
【美国加州拉荷亚2013年7月13日讯】由美国Scripps研究所引领的国际研究团队最近成功解析了人胰高血糖素受体的三维原子结构。胰高血糖素受体主要分布在肝脏和肾脏细胞上，参与调节体内血糖水平，是治疗2型糖尿病的潜在药物靶点。
“Our data should change the current view of how drugs are designed with this and related receptors,” said TSRI Research Associate Fai Yiu Siu, PhD, who was first author of the study.
The study is reported on July 17, 2013, in an advance online edition of the journal Nature, alongside a British laboratory’s structural study of another member of the same class of receptors—known as “class B” G protein-coupled receptors (GPCRs).
“我们的研究成果将会改变目前针对这一受体的药物设计思路。”这项研究的第一完成人、Scripps研究所研究助理Fai Yiu Siu博士说。相关成果已于2013年7月17日在《自然》杂志在线发表。同期《自然》还发表了由英国科学家解析的另一个B型G蛋白偶联受体的分子结构。
"Understanding how the glucagon receptor interacts with and binds to its partners will provide new information on how cells maintain sugar levels, possibly aiding the development of treatments for glucose-related disorders like type 2 diabetes," said Jean Chin, PhD, of the National Institutes of Health’s National Institute of General Medical Sciences, which partially funded the research. "Because the receptor is the first in its class of membrane proteins to be structurally determined, the work may advance studies of similarly shaped, medically important but often difficult to characterize molecules."
美国国立卫生研究院（NIH）对这项研究提供了部分资助，其下属的国立综合医学研究所的Jean Chin博士说：“阐明胰高血糖素与其受体的结合及作用方式将会为细胞如何维持血糖稳态提供新的信息，并将有助于开发治疗高血糖症（如2型糖尿病）的新方法。因为是同家族中第一个被解析结构的膜蛋白受体，这一成果对研究那些结构相似、医学意义重要但难以揭示的受体分子具有推进作用”。
TSRI Professor Raymond C. Stevens, PhD, who was a senior author of the study, noted, “This work involved a very fruitful international collaboration in which researchers in the United States, China and Europe worked closely together for more than two years to uncover the key differences in this subfamily of GPCRs.”
作为通讯作者之一的Scripps研究所教授Raymond C. Stevens博士着重指出：“该项由来自美国、中国和欧洲等地的科学家经过两年多的紧密合作所取得的成果发现了这个G蛋白偶联受体亚家族的重要特征。”
Popular Drug Targets
熟悉的药物靶点
GPCRs are the largest family of cellular receptors in humans and other animals. More than a third of all modern pharmaceuticals target these receptors, either to boost or block their activities. Determining the structural details of individual GPCRs and how they interact with binding partners has thus been a major goal of much biological research.
G蛋白偶联受体是一类存在于人类和动物细胞表面最大的受体家族。超过三分之一的现代药物是以G蛋白偶联受体为靶点，通过激动或抑制受体活性发挥疗效的。因此，阐明各种G蛋白偶联受体的确切结构及其与配体相互作用之机制乃是生物学研究的一个主要目标。
Over the past decade, the Stevens and Cherezov laboratories at TSRI have pioneered techniques to express, stabilize and induce these inherently flexible receptor proteins to form regular crystalline solids—from which their structures can be derived using a technique called X-ray crystallography. The crystal structures the team has “determined” so far include the groundbreaking crystallization and structure determination work of the b2 adrenergic receptor, followed by the A2A adenosine receptor (active and inactive state), S1P1 receptor, CXCR4 chemokine receptor, D3 dopamine receptor, histamine H1 receptor, kappa opioid receptor, nociception receptor, serotonin receptors 5HT1b and 5HT2b, and the smoothened receptor (a class F receptor)—all well-known and important therapeutic targets to treat a variety of human diseases. Each of these receptor systems is being followed up by careful structure-function studies including NMR in collaboration with the TSRI Wüthrich laboratory to fully understand how they work.
在过去的十多年中，Scripps研究所的Stevens和Cherezov实验室开创性地建立了一系列解析蛋白结构的研究手段，包括表达、稳定和诱导柔性蛋白形成规则的晶体，从而用X-线衍射技术分析其结构等。迄今为止，他们已经成功解析了多个G蛋白偶联受体的结构，包括b2肾上腺素受体、A2A腺苷酸受体（静止态和激活态）、D3多巴胺受体、组胺H1受体、k阿片受体、孤菲肽受体、五羟色胺5HT1b和五羟色胺5HT2b受体以及跨膜蛋白Smoothened受体（G蛋白偶联受体F家族成员）等。这些都是为人熟知且与多种疾病密切相关的重要靶点。目前，Stevens和Wüthrich两个实验室正在利用核磁共振技术对这些受体的结构和功能开展合作研究。
Other than the class F smoothened receptor, all the GPCRs whose structures have been solved to date are known as “class A” GPCRs for their common structural and protein-sequence features. More challenging for structural biologists has been the class B GPCRs, which include the glucagon receptor as well as several closely related protein molecules.
除了跨膜蛋白Smoothened受体外，已经解析出的所有G蛋白偶联受体均属于A家族，它们具有相近的结构和氨基酸序列特征。然而，包括胰高血糖素受体和几个相关蛋白分子在内的B家族受体给结构生物学家带来了更多的挑战。
“These receptors are very different from the class A receptors since they have key functional domains both embedded in and outside of the cellular membrane, so the lessons we’ve learned from the class A have not been entirely applicable,” said Vsevolod Katritch, an assistant professor of molecular biology at TSRI and a co-author on the study.
“这类受体与A家族成员相比存在很大的差别。由于它们的关键功能区域不仅暴露于表面而且包埋在细胞膜内，我们对A家族受体结构的认识并不完全适应于此”。Scripps研究所助理教授和该论文的共同作者之一Vsevolod Katritch博士有感而发。
Potential Way to Control Blood Sugar
控制血糖的潜在方法
The glucagon receptor and related GLP-1 and GIP receptors were high on the list of desired class B GPCR structures due to their potential as a drug targets for diabetes and their similarity to other receptors involved in endocrine and metabolic disorders. When activated by the hormone glucagon during fasting, the glucagon receptor triggers the release into the bloodstream of stored glucose from the liver and other sites. Modulating the glucagon receptor’s activity thus offers a way to control blood sugar.
由于胰高血糖素受体和相关的胰高血糖素样肽-1受体及葡萄糖依赖性胰岛素释放肽受体不仅是潜在的糖尿病治疗药物靶点而且与其它参与内分泌疾病和代谢紊乱进程的受体具有相似性，它们因此成为人们迫切期待解析结构的B族G蛋白偶联受体。在饥饿状态下，胰高血糖素激活胰高血糖素受体进而促进肝糖元向血液中释放葡萄糖，故调节胰高血糖素受体的活性是控制血糖的一条途径。
Other laboratories have reported finding the crystal structure of the small soluble part of the glucagon receptor, known as the extracellular domain. But the structure of the receptor’s midsection, normally anchored in the host cell’s membrane where the signal is transmitted, has been elusive. Although it is also involved in binding to glucagon, this “transmembrane domain” of the receptor—which resembles a jumble of 7 loose springs—tends to resist the crystallization that is needed for structure determination.
胰高血糖素受体胞外可溶区域的晶体结构已有报道，但对于锚定在细胞膜上负责信号转导的跨膜区域之结构仍不明确。胰高血糖素受体跨膜区域类似于7根松散的弹簧，虽然参与了受体结合，但因不易结晶从而使结构解析变得异常困难。
After many attempts, Siu was finally able to obtain crystallography-worthy crystals of the receptor’s transmembrane domain. Borrowing one key innovation from class A GPCR studies, he used a special “fusion protein” to hold the molecule together. The resulting structure, determined to a resolution of 3.4 Angstroms, turned out to have two key features that differ from those seen in class A GPCRs. One is an unusually elongated, stalk-like segment that connects the transmembrane region to the outermost, knob-like domain of the receptor. The other is an unusually large “pocket” within the transmembrane region where the N-terminal part of the glucagon peptide is expected to dock.
经过许多次艰难的尝试，Siu最终获得了胰高血糖素受体跨膜区域可用于结构解析的晶体。他借鉴了研究A族受体的关键创新技术即应用融合蛋白稳定经配体结合的受体，最终解析的分辨率为3.4埃的受体结构与A族受体有两个显著的差异。首先，其第一跨膜螺旋向细胞膜外延伸出3个α螺旋，形成“茎”样结构，用以“捕捉”胰高血糖素，使其氨基端插入跨膜区而与受体结合。其次，位于跨膜区域的配体结合“口袋”异常之大， 便于与胰高血糖素的氨基端相结合。
“If you’re trying to get a drug molecule to fit snugly into that pocket, you might need a larger one than those that are normally used to target class A GPCRs,” Siu said. He added that several pharmaceutical companies have been trying to develop drugs that specifically target this and related receptors. “Other than peptides, maybe the drugs they’re designing need to be bigger and not conform to the usual characteristics of other drugs.”
“如果想得到一个与结合“口袋”紧密契合的药物，你可能需要一个比通常靶向A族受体更大的分子”。Siu还指出，已有数家药企尝试开发针对胰高血糖素受体及其相关受体的药物，“除了多肽，他们也许需要设计出不同于传统药物特征、分子更大的新药。”
Putting Together Pieces of the Puzzle
谜团的成功破解
One of the other tour de forces of the study included extensive analyses of how the receptor’s glucagon-binding properties change when its individual amino acids are mutated. This work, involving the study of more than 100 separate mutations to the receptor, was performed by co-senior author Ming-Wei Wang’s laboratory at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Chris de Graaf’s laboratory at the Amsterdam Institute for Molecules, Medicines and Systems (AIMMS) of VU University Amsterdam, and Jesper Lau at Novo Nordisk.
这项研究的另一个亮点是应用突变技术，深入分析了该受体经100多个氨基酸单独突变后与胰高血糖素结合活力的改变。这部分工作是由论文的另一位通讯作者中国科学院上海药物研究所王明伟课题组、荷兰阿姆斯特丹自由大学分子药物系统实验室的Chris de Graaf博士以及丹麦诺和诺德公司的Jesper Lau博士共同完成的。
Combining the new data with previous work, computational biochemists De Graaf and Katritch were able to develop a detailed model for how the full-length glucagon receptor operates: its outermost domain grabs one end of the glucagon peptide, then inserts the other end of the peptide into the large binding pocket in the transmembrane domain, locking the receptor structure in place and triggering receptor activation. “We managed to put together the pieces of this puzzle, and the mutational analysis was a key part of our model,” said Stevens.
Siu, Katritch, Stevens and their colleagues now are trying to determine the glucagon receptor structure while the receptor is bound to glucagon, and at a sharper resolution.
在将新获数据和既往信息整合后，计算生物学家De Graaf 博士和 Katritch博士构建了胰高血糖素受体的全长模型：受体伸出膜外的最突出部分“钳住”了胰高血糖素分子的一端，然后将另一端插入受体跨膜区域内又大又深的结合“口袋”中。这样胰高血糖素就可以“锁定”受体结构进而激动其活性。“在把琐碎的信息搭拼整合后，突变分析的结论是这个模型的关键部分。”Stevens教授说。目前，Siu、Katritch、Stevens和他们的同事们正在培养胰高血糖素与其受体相结合的晶体，以期得到更为精准的结构信息。
“This glucagon receptor structure has opened the door to understanding how hormone peptides bind to this class of receptors, and it will help us solve related receptor structures,” Stevens said. “Our knowledge of GPCRs is still in its infancy, and we are learning a great deal with each new structure, with different techniques and in the different functional states.”
“胰高血糖素受体结构的解析给我们打开了一扇理解多肽激素如何与B族受体结合的大门，它将帮助我们解析更多相关受体的结构。”Stevens说道。“我们对G蛋白偶联受体的认识尚处在初级阶段，每次采用不同的技术、在不同的功能状态下解析出一个新结构都使我们受益匪浅。”
The other contributors to the study, “Structure of the human glucagon class B G-protein-coupled receptor,” were Min He, Dehua Yang, Zhiyun Zhang and Caihong Zhou of Chinese Academy of Sciences; Gye Won Han, Daniel Wacker, Jeremiah S. Joseph, Wei Liu and Vadim Cherezov of TSRI; Qingping Xu of the SLAC National Accelerator Laboratory; and Jesper Lau of Novo Nordisk in Denmark.
《G蛋白偶联受体B家族成员人胰高血糖素受体的结构》论文的其他作者包括：中国科学院上海药物研究所的何敏、杨德华、张志云和周彩红；Scripps研究所的Gye Won Han、Daniel Wacker、Jeremiah S. Joseph、Wei Liu和 Vadim Cherezov；美国SLAC国家加速器实验室的Qingping Xu和丹麦诺和诺德公司的Jesper Lau等。
The study was supported by the National Institutes of Health (P50 GM073197, U54 GM094618, F32 DK088392); the Chinese Ministry of Health (2012ZX09304-011, 2013ZX09507002); the Shanghai Science and Technology Development Fund (11DZ2292200); the Novo Nordisk-Chinese Academy of Sciences Research Fund (NNCAS-2011-7); the Thousand Talents Program in China; and the Netherlands Organization for Scientific Research through a VENI grant (700.59.408).
这项研究分别获得了美国国立卫生研究院、中国卫生部、上海市科委，诺和诺德¾-中国科学院研究基金、中国“千人计划”项目以及荷兰科学研究组织VENI基金等的共同资助。
