Determining how the giant Streptococcus Pneumoniae IgA1 protease cleaves its host IgA1 substrate and how this interaction can be blocked

项目摘要 肺炎链球菌（SPN）是肺炎患者社区获得性肺炎的主要病原体， 在美国和欧洲，仍然是儿童细菌性肺炎和脑膜炎的主要原因 据世界卫生组织称，这是一个“重大的全球公共卫生问题”。而 目前的疫苗靶向仅包含已知血清型的子集的表面多糖， 针对更广泛表达的蛋白质毒力因子，如SPN IgA 1 蛋白酶（IgA 1 P）正在研究中。然而，SPN IgA 1 P和其IgA 1的相对大的尺寸， 它切割的底物阻碍了最初的宿主免疫反应，以前排除了旨在 阐明其分子结构和相互作用。通过综合开发新的策略， 结合多种生物物理/生物化学方法，包括冷冻EM，我们已经开始 阐明了SPNIgA 1 P的结构。值得注意的是，尽管存在新的结构折叠，SPN 在结构域之间形成的IgA 1 P活性位点与其他Zn-金属蛋白酶相同，并证实了我们的发现。 之前发表的催化机制。考虑到最近在利用基于结构的战略方面取得的成功， 为了开发基础广泛的疫苗，我们的目标是提供第一个高分辨率的结构， 单独的IgA 1 P，连同其IgA 1底物，以及与第一代单克隆抗体（mAb）， 阻断IgA 1 P活性。这些研究将对我们如何生产疫苗产生重大影响， 通过靶向IgA 1 P阻断SPN感染的治疗剂。 假设：大SPN IgA 1 P包含多个独立折叠的亚基，形成独特的3- 三维结构，其包括连接以形成金属蛋白酶活性位点的亚基，以适当地 使IgA 1定向以进行铰链切割。此外，该活性位点被中和mAb封闭。我们将 通过以下具体目标解决这一假设： 目的1）完成新型IgA 1 P催化区的冷冻EM结构，以确定其如何 亚基相互作用形成活性金属蛋白酶。 目的2）确定IgA 1 P如何与其IgA 1底物相互作用，以及这种相互作用如何被一种 单克隆抗体

PROJECT SUMMARY Streptococcus pneumoniae (SPN) is the primary causative agent for community-acquired pneumonia in the United States and Europe and remains the leading cause of bacterial pneumonia and meningitis in children worldwide, making it a “major global public health problem” according to the World Health Organization. While current vaccines target surface polysaccharides that comprise only a subset of the known serotypes, strategies aimed against a more widely expressed protein virulence factor have been advocated, such as the SPN IgA1 Protease (IgA1P) under investigation here. However, the relatively large size of SPN IgA1P and its IgA1 substrate that it cleaves to thwart the initial host immune response has previously precluded studies aimed at elucidating its molecular structure and interactions. By developing novel strategies through an integrative approach that combines multiple biophysical/biochemical methods including cryo-EM, we have begun elucidating the structure of SPN IgA1P. Remarkably, despite the presence of novel structural folds, the SPN IgA1P active site that is formed between domains is identical to other Zn-metalloproteases and confirms our previously published catalytic mechanism. Considering recent success in utilizing structure-based strategies for the development of broad-based vaccines, our goals here are to provide the first high-resolution structures of IgA1P alone, together with its IgA1 substrate, and with a first-generation monoclonal antibody (mAb) that blocks IgA1P activity. These studies will have a major impact on how we will generate vaccines and potentially therapeutics to block SPN infection through targeting IgA1P. Hypothesis: The large SPN IgA1P comprises multiple independently folded subunits that forms a unique 3- dimensional structure, which includes subunits that join to form the metalloprotease active site to properly orient the IgA1 for hinge cleavage. Furthermore, this active site is occluded by a neutralizing mAb. We will address this hypothesis through the following specific aims: Aim 1) Complete the cryo-EM structure of the novel IgA1P catalytic region to determine how its subunits interact to form the active metalloprotease. Aim 2) Determine how IgA1P interacts with its IgA1 substrate and how this interaction is blocked by a monoclonal antibody.