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: 10316789
• 负责人: Karen N. Allen
• 金额: $ 45.13万
• 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/10316789
• 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/antagonist; 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.

复合糖结合物在细菌的生存、定植和毒力中起着关键作用 共生菌和病原菌与它们的人类宿主之间的相互作用。一个重要的 这些结构的组装机制在细胞膜的细胞质表面上启动， 由聚戊烯醇磷酸(PrenP)磷酸糖基转移酶(PGTS)催化。PGTS传输一个c1‘- 来自可溶性二磷酸核苷(NDP)活化供体的磷糖与PrenP受体结合，产生 一种膜结合的聚戊烯醇二磷糖。我们的研究集中在一个具有单调性的PGT超家族 膜拓扑(MonPGTS)，直到我们最近的研究，对于它，只有有限的结构 和机械信息。这些酶不同于众所周知的多面体PGTS(PolyPGTS)，后者 具有许多跨膜序列。弯曲菌的生化研究和结构 Contenisus PglC，表明单PGT包括一个仅穿透的可再入膜螺旋(RMH) 双层的一张传单，然后重新出现。这项计划将追求协同生化，生物信息学， 单一PGTS的结构和化学生物学研究。在目标1中，结构将通过X射线确定 与洗涤剂溶解的蛋白质的结晶学，以及在膜环境中通过增溶到 脂类纳米颗粒和脂类立方相中的结晶。冷冻-EM中的脂质纳米粒也将 追求最佳体型的成员。与底物和抑制剂连接的结构和活性 分析，我们将阐明新发现的单PGT的特异性决定因素，并提供 关于它们在各种病原体的糖共轭生物合成途径中的作用的信息。在目标2中， 模拟UDP-糖底物的结合触发可溶性环的运动以完成 底物结合决定因素和关闭活性部位的催化，将使用交联剂和 在洗涤剂增溶和模型膜环境中基于荧光的方法。要提供 膜驻留的PrenP底物-RMH序列结合的补充洞察力 将通过信息学进行分析。这些信息将被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，用于预测无关蛋白质中的RMH 整个蛋白质组的家庭。AIM 3将开发核苷类似物作为抑制剂和 单调PGT超家族的基于活性的蛋白质谱探针。这一分析将定义 配基对结合和鉴定新的PGTS的贡献及其在细菌中的意义 代谢和宿主感染。最终，识别出的蛋白质可以作为发展的目标 新的抗菌和抗病毒药物。总体而言，这一深入研究的结构和约束 MonoPGT超家族的景观和生物探针的设计将奠定基础 确认和干预潜在治疗靶点的作用所需的知识和工具。