Structural Vaccinology of the Malaria Sporozoite Surface Sheath

描述（由申请人提供）：我们提出了一种创新的结构生物学方法，利用X射线晶体学和冷冻电子断层扫描的综合信息来刺激红细胞前疟疾疫苗和药物开发，以开发子孢子表面的详细分子形貌。这项工作将提供两个最关键的表面蛋白（CSP）和血小板反应蛋白相关的匿名蛋白（TRAP），以及它们在子孢子表面上最有效的表位的天然排列的三维地图。这项工作也将是重要的寄生虫运动，细胞入侵和免疫逃避的结构机制提供见解。长期目标是促进合理的疟疾疫苗开发和基于结构的药物设计，我们有三个具体目标。1.我们建议CSP包装在一个特定的方式，形成子孢子鞘。CSP包含N-末端、重复和血小板反应蛋白I型重复（TSR）结构域。我们将解决CSP的TSR结构域的晶体结构，这与其他蛋白质中的TSR结构域显著不同，在几个不同的晶格中。我们假设，我们将发现晶格，模仿包装的CSP TSR域的子孢子表面上。我们将解释的结构，包括一个假定的1-螺旋的存在下，在其功能支持稳定的，但灵活的，CSP分子之间的相互作用的子孢子表面。2.我们将解决的串联冯维勒布兰德因子A（VWA）和TSR域的TRAP的晶体结构。我们希望了解如何配体结合TRAP介导滑行运动和细胞侵袭。结构将检验VWA和TSR结构域以有利于通过VWA结构域和胞质结构域之间的张力传递的构象变化的方式彼此相互作用的假设。3.我们将应用冷冻电子断层扫描来确定子孢子表面的超微结构细节。我们期望看到三个鞘层对应的N-末端，重复，和TSR域，不同的电子密度和包装。我们希望能够使用subtomograms平均分析包装在最近膜层，并比较这种包装的TSR域的晶格。我们也可以在CSP的海洋中看到其他分子的岛屿，如TRAP。通过晶体学、冷冻电子断层扫描和显微镜的互补使用，我们计划构建子孢子鞘的模型。这些发现应该提供重要的见解表位是如何屏蔽或暴露在鞘在不同的时间在感染过程中，以及如何apicomplexans屏蔽自己的免疫系统。

DESCRIPTION (provided by applicant): We propose an innovative structural biology approach to stimulate pre-erythrocytic malaria vaccine and drug development using integrated information from X-ray crystallography and cryoelectron tomography to develop a detailed molecular topography of the sporozoite surface. This work will provide a three-dimensional map of the native arrangement of two of the most critical surface proteins, (CSP) and thrombospondin-related anonymous protein (TRAP), and their most potent epitopes on the sporozoite surface. This work will also be of significance for providing insights into the structural mechanisms of parasite motility, cell invasion, and immune evasion. With the long term goal of stimulating rational malaria vaccine development and structure-based drug design, we have three specific aims. 1. We propose that CSP packs in a specific way to form the sporozoite sheath. CSP contains N-terminal, repeat, and thrombospondin type I repeat (TSR) domains. We will solve the crystal structure of the TSR domain of CSP, which differs significantly from the TSR domain in other proteins, in several different lattices. We hypothesize that we will find crystal lattices that mimic packing of the CSP TSR domain on the sporozoite surface. We will interpret the structure, including the presence of a putative 1-helix, in terms of its function in supporting stable, yet flexible, interactions between CSP molecules on the sporozoite surface. 2. We will solve the crystal structure of the tandem von Willebrand factor A (VWA) and TSR domains in TRAP. We wish to understand how ligand binding to TRAP mediates gliding motility and cell invasion. Structures will test the hypothesis that the VWA and TSR domains interact with one another in a manner conducive to conformational change transmitted by tensile force between the VWA domain and the cytoplasmic domain. 3. We will apply cryoelectron tomography to define ultrastructural details of the sporozoite surface. We expect to see three sheath layers corresponding to the N-terminal, repeat, and TSR domains, that differ in electron density and in packing. We expect to be able to use subtomogram averaging to analyze packing in the most membrane-proximal layer, and to compare this packing to that of the TSR domain in crystal lattices. We may also see islands of other molecules such as TRAP in a sea of CSP. Through complementary use of crystallography and cryolectron tomography and microscopy we plan to construct a model of the sporozoite sheath. These findings should provide important insights into how epitopes are shielded or exposed in the sheath at different times in the infection process, and how apicomplexans shield themselves from the immune system.