In-depth structural characterization of the tetraspanin CD81

每个人体细胞都包裹着一层细胞膜，将细胞内容物与周围环境分开。嵌入这层膜的蛋白质就像大门一样，允许分子进出细胞；它们还调解细胞与其环境之间发生的相互作用。这意味着膜蛋白参与了正常细胞功能的许多最基本的过程；当这些过程失败时，疾病就会产生。因此，毫不奇怪，有史以来最畅销的十大小分子药物都是针对膜蛋白的。在任何给定的细胞中都有许多不同的膜蛋白，分为1500多个家族，每个家族都有许多成员。为了详细研究它们中的任何一种，了解它们的三维结构是很重要的。这项研究的核心是一种叫做x射线晶体学的技术，它使科学家能够获得蛋白质内部原子如何排列的详细视图，为进一步研究提供框架。科学家们利用这个框架来研究蛋白质的功能，使人们对细胞在健康和疾病中的作用有了新的认识，并为开发新药提供了知识。四联蛋白是一种膜蛋白，通过与多种其他膜蛋白和可溶性蛋白相互作用而发挥作用，从而影响细胞的信号传递、相互作用、形状改变和运动。值得注意的是，四联蛋白还参与了多种疾病的感染过程。然而，由于没有任何已知的全长四蛋白家族成员的结构，四蛋白在这些基本过程中的作用方式尚不清楚，这给我们的细胞生物学知识留下了重大空白。获得任何膜蛋白的结构都是一项重大的科学挑战：必须从细胞膜上去除蛋白质，这通常会导致蛋白质变得非常不稳定，以至于无法用于制作x射线晶体学所需的晶体。因此，我们对许多具有重要生物学功能的膜蛋白家族知之甚少。我们现在已经克服了四联蛋白CD81的结晶挑战。人类CD81是四联蛋白家族成员之一，也是我们拟研究的主题。它在细胞相互作用、免疫反应和受精中发挥着重要作用。值得注意的是，CD81是一些非常重要的人类病原体的受体，包括流感、人类免疫缺陷病毒、疟疾寄生虫、1型嗜t淋巴细胞病毒和丙型肝炎病毒（HCV）。它也可能是肿瘤启动子。CD81的核心功能（以及所有四联蛋白的核心功能）是它与自身和其他蛋白质形成广泛相互作用的能力；然而，如上所述，我们不知道这些结构是什么样子的，因此缺乏进一步研究的框架。我们在提案中概述的研究的第一个目标是解决CD81的三维结构。我们已经在这个目标上取得了很好的进展，已经结晶了CD81并收集了x射线衍射数据。我们还与法国的科学家合作，他们可以制造结合CD81的HCV蛋白E2的可溶性形式。我们项目的第二个目标是制造一个HCV-E2/CD81复合物，这样我们就可以表征它并解决它的结构；这将使我们更多地了解CD81如何与其他蛋白质相互作用。我们相信，我们是世界上唯一拥有应对这一挑战的所有工具的团队。结构生物学的全新发展（例如，高分辨率电子显微镜）使我们能够设计第三个目标，即观察细胞膜中的这些结构（通过电子断层扫描），将我们的原子水平结构数据与细胞中实际发生的事情联系起来。在这种水平上详细研究CD81的结构将使我们开始了解四跨蛋白如何在健康和疾病中起作用。

Every human cell is encased by a cell membrane that separates the cell contents from its surroundings. Proteins embedded in this membrane act as gates to allow molecules to enter and exit cells; they also mediate the interactions that occur between a cell and its environment. This means that membrane proteins are involved in many of the most fundamental processes in normal cell function; when these processes fail, diseases result. It is no surprise, then, that the top ten best-selling small molecule drugs of all time all target membrane proteins.There are many different membrane proteins in any given cell, grouped into over 1,500 families, each with many members. In order to study any of them in detail, it is important to understand their three-dimensional structures. Central to this is a technique called X-ray crystallography that allows scientists to obtain a detailed view of how the atoms within a protein are arranged, providing a framework for further study. Scientists use this framework to investigate how the protein functions, bringing new levels of understanding to how cells work in health and disease, and providing knowledge to develop new drugs.Tetraspanins are membrane proteins that function by interacting with a wide range of other membrane and soluble proteins, thereby affecting how cells signal, interact, change shape and move. Remarkably, tetraspanins are also involved in the process of infection for a wide range of diseases. However, because there is no known structure of any full-length tetraspanin family member, the mode of action of tetraspanins in these essential processes is not understood, leaving a major gap in our knowledge of cell biology.Obtaining the structure of any membrane protein is a major scientific challenge: It is necessary to remove the protein from the cell membrane which often results in the protein becoming so unstable that it cannot be used to make the crystals required to perform X-ray crystallography. Consequently, we know very little about many membrane protein families with important biological functions. We have now overcome this crystallization challenge for the tetraspanin, CD81.Human CD81 is one of the best understood tetraspanin family members and is the subject of our proposed research. It has well-established roles in how cells interact with each other, the immune response and fertilization. Notably CD81 is a receptor for some very important human pathogens including influenza, human immunodeficiency virus, the malarial parasite, T-cell lymphotropic virus type 1 and hepatitis C virus (HCV). It may also be a tumour promoter. Central to CD81 function (and to that of all tetraspanins) is its ability to form extensive interactions with itself and other proteins; however, we don't know what these structures look like and therefore lack the framework for further study, mentioned above.The first aim of the research outlined in our proposal is to solve the three-dimensional structure of CD81. We have made excellent progress towards this goal, having crystallized CD81 and collected X-ray diffraction data.We have also teamed up with scientists in France who can make soluble forms of the HCV protein, E2, that binds CD81. The second aim of our project is to make an HCV-E2/CD81 complex so we can characterize it and solve its structure; this will allow us to learn more about how CD81 interacts with other proteins. We believe we are the only team in the world that has all the tools to take on this challenge.Brand new developments in structural biology (e.g. high-resolution electron microscopy) have enabled us to devise a third aim, which is to look at these structures in the cell membrane (by electron tomography), linking our atomic level structural data to what is actually happening in the cell.Studying the structure of CD81 at this level of detail will allow us to begin to understand how tetraspanins work in health and disease.