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的生存、定植和毒力中发挥着关键作用，并有助于 共生细菌和致病细菌与其人类宿主之间的相互作用。 这些结构的组装机制是在细胞膜的细胞质面上启动的， 由聚戊烯醇磷酸 (PrenP) 磷酸糖基转移酶 (PGT) 催化转移 C1'-。 磷酸糖从可溶性二磷酸核苷 (NDP) 激活的供体转移到 PrenP 受体，产生 我们的研究重点是具有单位的 PGT 超家族。 膜拓扑（monoPGT），直到我们最近的研究为止，只有有限的结构 这些酶与众所周知的多位PGT (polyPGT) 不同。 具有许多跨膜序列和弯曲杆菌的结构。 concisus PglC，表明 monoPGT 包含仅穿透的可重入膜螺旋 (RMH) 双层的一张传单，然后重新出现，该计划将追求协同生物化学、生物信息学、 In Aim 1 结构的结构和化学生物学研究将通过 X 射线确定。 使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成 脂质纳米颗粒和纳米脂质颗粒中的冷冻电镜结晶也将被研究。 寻找最佳尺寸的成员以及底物和抑制剂的配体结构和活性。 分析后，我们将阐明新鉴定的 monoPGT 的特异性决定因素，并提供 关于它们在各种病原体的糖复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环的移动以完成的模型 底物结合决定簇并关闭催化活性位点，将使用交联和 去垢剂溶解和模型膜环境中基于荧光的方法。 对驻留在膜上的 PrenP 底物、RMH 序列的结合的补充见解 将通过信息学分析该信息将用于开发隐马尔可夫模型来识别。 单位 PGT 超家族中的 RMH 片段，用于预测不相关蛋白质中的 RMH Aim 3 将开发核苷类似物作为抑制剂和 该分析将定义单位 PGT 超家族的基于活性的蛋白质分析探针。 配体部分对结合和识别新 PGT 的贡献及其在细菌中的意义 最终，所鉴定的蛋白质可以作为发展的靶标。 总体而言，这种新型抗菌和抗毒剂的结构和结合进行了深入研究。 monoPGT 超家族的景观和生物探针的设计将建立基础 验证和干预潜在治疗目标的作用所需的知识和工具。