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来刺激红细胞前疟疾疫苗和药物的开发，利用来自X射线结晶学和冷冻电子断层扫描的综合信息来开发子孢子表面的详细分子拓扑图。这项工作将提供两个最关键的表面蛋白(CSP)和凝血酶相关匿名蛋白(TRAP)的天然排列及其在子孢子表面最有效的表位的三维图谱。这项工作也将对深入了解寄生虫运动、细胞入侵和免疫逃避的结构机制具有重要意义。我们的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.我们提出CSP以一种特定的方式包装形成子孢子鞘。CSP含有N-末端、重复和凝血酶反应蛋白I型重复(TSR)结构域。我们将解决CSP的TSR结构域在几个不同的晶格中的晶体结构，它与其他蛋白质中的TSR结构域有很大的不同。我们假设我们会找到模拟CSP TSR结构域在子孢子表面的填充的晶格。我们将解释这种结构，包括假定的1-螺旋的存在，根据它在支持子孢子表面CSP分子之间稳定而灵活的相互作用方面的功能。2.解决了陷阱中串联von Willebrand因子A(VWA)和TSR结构域的晶体结构。我们希望了解与TRAP结合的配体是如何介导滑动运动和细胞侵袭的。结构将检验这一假设，即VWA和TSR结构域以一种有利于通过张力在VWA结构域和细胞质结构域之间传递的构象变化的方式相互作用。3.我们将应用低温电子断层扫描技术来确定子孢子表面的超微结构细节。我们预计会看到与N-末端、重复和TSR结构域相对应的三层鞘，它们在电子密度和堆积方面有所不同。我们期望能够使用亚断层图像平均来分析膜最近端的堆积，并将这种堆积与晶体晶格中的TSR结构域的堆积进行比较。我们也可能看到其他分子的岛屿，例如CSP海洋中的陷阱。通过补充使用结晶学和冷冻电子断层扫描和显微镜，我们计划构建一个子孢子鞘的模型。这些发现应该为以下方面提供重要的见解：在感染过程的不同时间，表位是如何被屏蔽或暴露在鞘中的，以及顶端复合体如何保护自己免受免疫系统的攻击。

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.