Structure and function of the monotopic phosphoglycosyl transferase superfamily: Initiators of biosynthesis of complex bacterial glycoconjugates

复杂的糖缀合物在细菌的存活、定殖和毒力中起关键作用，并且有助于细菌的存活、定殖和毒力。 共生和致病细菌与人类宿主之间的相互作用。一个重要 这些结构的组装机制起始于细胞膜的细胞质表面， 由磷酸聚戊烯醇（PrenP）磷酸糖基转移酶（PGTs）催化。PGTs转移C1 '- 磷酸糖从可溶性核苷二磷酸（NDP）活化的供体转化为PrenP受体， 一种膜结合的聚戊烯醇二磷酸糖。我们的研究集中在一个PGT超家族， 膜拓扑结构（monoPGTs），直到我们最近的研究，只有有限的结构 机械信息。这些酶不同于众所周知的多位PGTs（polyPGTs），其 具有许多跨膜序列。弯曲杆菌的生化研究和结构 concisus PglC，表明monoPGTs包括一个只穿透 双层的一个小叶，然后重新出现。该计划将追求协同生物化学，生物信息学， monoPGTs的结构和化学生物学研究。在目标1中，将通过X射线确定结构 用洗涤剂溶解的蛋白质进行晶体学分析，并在膜环境中，通过溶解到 脂质纳米颗粒和在无规立方相中结晶。脂质纳米颗粒中的Cryo-EM也将被 追求最佳规模的成员。连同底物和抑制剂配体结构和活性 分析，我们将阐明新确定的monoPGTs的特异性决定因素，并提供 关于它们在各种病原体的糖缀合物生物合成途径中的功能的信息。在目标2中， UDP-糖底物的结合触发可溶性环的运动以完成 底物结合决定簇和关闭催化活性位点，将使用交联和 基于荧光的方法在洗涤剂溶解和模型膜环境。提供 互补的洞察力结合的膜居民PrenP底物，RMH序列 将通过信息学进行分析。这些信息将用于开发隐马尔可夫模型，以识别 单调PGT超家族中的RMH片段，用于预测不相关蛋白质中的RMH 蛋白质组中的家族。Aim 3将开发核苷类似物作为抑制剂， 基于活性的蛋白质分析探针的单调PGT超家族。该分析将定义 配体部分对结合和鉴定新PGTs的贡献及其在细菌中的意义 代谢和宿主感染。最终，所鉴定的蛋白质可以作为发展的靶点， 新的抗菌剂和抗病毒剂。总的来说，本文深入研究了 monoPGT超家族的景观和生物探针的设计将建立基本的 验证和干预潜在治疗靶点作用所需的知识和工具。

Complex glycoconjugates play a pivotal role in bacterial survival, colonization and virulence and contribute to the interactions between symbiotic and pathogenic bacteria and their human hosts. An important mechanism for the assembly of these structures is initiated on the cytoplasmic face of cell membranes, catalyzed by polyprenol phosphate (PrenP) phosphoglycosyl transferases (PGTs). PGTs transfer a C1’- phosphosugar from a soluble nucleoside diphosphate (NDP) activated donor to a PrenP acceptor, yielding a membrane-bound polyprenol diphosphosugar. Our studies focus on a PGT superfamily with a monotopic membrane topology (monoPGTs) for which, until our recent studies, there has been only limited structural and mechanistic information. These enzymes differ from the well-known polytopic PGTs (polyPGTs), which bear many membrane-spanning sequences. Biochemical studies and the structure of Campylobacter concisus PglC, show that the monoPGTs include a reentrant membrane helix (RMH) that penetrates only one leaflet of the bilayer, then re-emerges. This program will pursue synergistic biochemical, bioinformatic, structural and chemical biology studies of the monoPGTs. In Aim 1 structures will be determined via X-ray crystallography with detergent-solubilized protein and, in a membrane environment, by solubilization into lipid nanoparticles and crystallization in the lipidic cubic phase. Cryo-EM in lipid nanoparticles will also be pursued for members of optimal size. Together with substrate and inhibitor liganded structures and activity analysis, we will elucidate the specificity determinants of newly-identified monoPGTs and provide information on their function in the glycoconjugate biosynthetic pathways of various pathogens. In Aim 2, the model that binding of the UDP-sugar substrate triggers the movement of a soluble loop to complete substrate-binding determinants and close the active site for catalysis, will be tested using cross-linking and fluorescence-based approaches in detergent-solubilized and model membrane environments. To provide complementary insight into the binding of the membrane-resident PrenP substrate, the RMH sequences will be analyzed via informatics. This information will be used to develop hidden Markov models to identify RMH segments within the monotopic PGT superfamily and used to predict RMHs in unrelated proteins families across the proteome. Aim 3 will develop nucleoside analogs that will serve as inhibitors and activity-based protein profiling probes of the monotopic PGT superfamily. This analysis will define the contribution of ligand moieties to binding and identify new PGTs and their significance in bacterial metabolism and host infection. Ultimately, the identified proteins can act as targets for the development of new antibacterial and antivirulence agents. Overall, this in-depth study of the structures and binding landscape of the monoPGT superfamily and design of biological probes will establish the fundamental knowledge and tools needed for validating and intervening in the action of potential therapeutic targets.