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

单位磷酸糖基转移酶超家族的结构和功能：复杂细菌糖复合物生物合成的引发剂

• 批准号: 10447209
• 负责人: Karen N. Allen
• 金额: $ 43.76万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447209
• 关键词: Active Sites; Anabolism; Anti-Bacterial Agents; Bacteria; Behavior; Binding; Biochemical; Bioinformatics; Biological; Biological Models; Biology; Campylobacter; Campylobacter jejuni; Catalysis; Cell membrane; Cells; Cellular Membrane; Chemicals; Complex; Cryoelectron Microscopy; Crystallization; Cysteine; Dependence; Detergents; Development; Environment; Enzymes; Epitopes; Face; Family; Fluorescence; Foundations; Glycoconjugates; Human; Infection; Informatics; Integral Membrane Protein; Knowledge; Label; Lead; Ligands; Lipids; Membrane; Modeling; Molecular; Molecular Conformation; Molecular Structure; Movement; Pathogenesis; Pathway interactions; Phase; Play; Polysaccharides; Process; Protein Family; Proteins; Proteome; Ribose; Roentgen Rays; Role; Scaffolding Protein; Solid; Specificity; Structure; Substrate Specificity; Testing; Therapeutic; Transferase; Uridine; Uridine Diphosphate Sugars; Ursidae Family; Virulence; X-Ray Crystallography; activity-based protein profiling; bacterial metabolism; base; biophysical analysis; cell envelope; conformer; crosslink; defined contribution; design; flexibility; human pathogen; inhibitor; inorganic phosphate; insight; lipid nanoparticle; markov model; member; membrane model; molecular dynamics; nucleoside analog; nucleoside diphosphate; pathogen; pathogenic bacteria; programs; protein folding; scaffold; small molecule; structural biology; sugar; sugar nucleotide; symbiont; therapeutic target; tool

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.

复杂的糖缀合物在细菌存活，定殖和病毒中起关键作用，并有助于 共生和致病细菌与其人类宿主之间的相互作用。一个重要的 这些结构组装的机制是在细胞膜的细胞质面上启动的， 由磷酸聚苯酚（PRENP）磷酸糖基转递（PGTS）催化。 pgts转移c1'-- 来自可溶性核苷二磷酸（NDP）激活供体的磷果糖向PRENP受体，产生 膜结合的多发性双磷酶。我们的研究专注于具有单位型的PGT超家族 膜拓扑（monopgts），直到我们最近的研究才有有限的结构性 和机械信息。这些酶不同于众所周知的多重PGT（polypgts），这些酶 带有许多跨膜序列。生化研究和弯曲杆菌的结构 Concisus PGLC，表明单体包括仅穿透只穿透的膜螺旋（RMH） 双层的一个传单，然后重新出口。该程序将纯化协同生物化学，生物信息学， 单体的结构和化学生物学研究。在AIM 1中将通过X射线确定 用洗涤剂 - 溶解蛋白和在膜环境中的晶体学，通过溶解 脂质纳米颗粒和脂质立方相结晶。脂质纳米颗粒中的冷冻EM也将是 追求最佳大小的成员。与底物和抑制剂配体结构和活性一起 分析，我们将阐明新识别的单体的特异性确定剂，并提供 有关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AIM 2中， UDP-SUGAR底物结合的模型触发可溶循环的运动 底物结合确定并关闭活性位点进行催化，将使用交联和 确定溶解化和模型膜环境中的基于荧光的方法。提供 完全深入了解膜居民PRENP底物的结合，RMH序列 将通过信息分析。此信息将用于开发隐藏的马尔可夫模型以识别 单位PGT超家族中的RMH段，用于预测无关蛋白质的RMH 跨蛋白质组的家庭。 AIM 3将发展为抑制剂的核外侧类似物， 基于活性的蛋白质分析问题是单位PGT超家族的。该分析将定义 配体部分对结合和识别新PGT的贡献及其在细菌中的重要性 代谢和宿主感染。最终，确定的蛋白质可以充当开发的目标 新的抗菌和抗动力剂。总体而言，这项对结构和结合的深入研究 Monopgt超家族的景观和生物学问题的设计将确定基本 验证和干预潜在治疗靶标的所需的知识和工具。