Conformational dynamics and allosteric regulation during stress-responsive metallocofactor assembly

应激反应性金属辅因子组装过程中的构象动力学和变构调节

• 批准号: 10380857
• 负责人: Patrick Frantom
• 金额: $ 29.86万
• 依托单位: UNIVERSITY OF ALABAMA IN TUSCALOOSA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-10 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380857
• 关键词: ATP phosphohydrolase; Affinity; Allosteric Regulation; Anti-Bacterial Agents; Antibiotics; Archaea; Bacteria; Binding; Biochemical; Biogenesis; Biological Assay; Biophysics; Carrier Proteins; Cells; Complement; Complex; Crystallography; Cysteine; Defect; Deuterium; Development; Dimerization; Docking; Emergency Situation; Enterococcus faecalis; Escherichia coli; Event; Funding; Genetic; Goals; Human; Hydrogen; Iron; Mass Spectrum Analysis; Modeling; Molecular Conformation; Multiprotein Complexes; Mycobacterium tuberculosis; Nature; Organism; Outcome; Oxidative Stress; Pathway interactions; Play; Process; Prokaryotic Cells; Proteins; Regulation; Research; Role; Site; Source; Starvation; Stress; Structural Models; Structure; Sulfides; Sulfur; System; Testing; base; cofactor; cysteine desulfurase; design; experimental study; in vivo; novel; novel therapeutic intervention; pathogen; pathogenic bacteria; persulfides; prevent; protein protein interaction; protein transport; rational design; scaffold; small molecule; trafficking

PROJECT SUMMARY Iron-sulfur (Fe-S) cofactor biogenesis is essential for most living organisms. The continuing long-term goal of this renewal proposal is to characterize protein-protein interactions (PPIs) critical for bacterial Fe-S cluster biogenesis by the Suf (sulfur formation) pathway, which is ubiquitous in prokaryotes including important pathogens. Due to the toxic nature of free iron and sulfide in cells, Fe-S cluster assembly and trafficking is highly choreographed by a complex network of protein partners. In Escherichia coli, Suf has evolved to act as an emergency pathway that is activated under conditions of oxidative stress or iron starvation. In other bacteria, such as Mycobacterium tuberculosis and Enterococcus faecalis, the Suf pathway is the sole source for Fe-S clusters, making it an essential pathway and potential target for antibiotic development. The Suf pathway consists of six proteins, SufABCDSE. SufS is a cysteine desulfurase that mobilizes persulfide (So) from L-cysteine. Persulfide generated by SufS must be transferred to the transpersulfurase protein, SufE, prior to delivery to SufB on the SufBC2D cluster scaffold. In vivo, Fe-S cluster assembly on the SufBC2D scaffold is dependent on the ATPase activity of SufC and acquisition of iron, which is likely regulated by SufD. Following cluster assembly, SufBC2D transfers the nascent cluster to SufA for downstream trafficking. While simple functional assignments are available for proteins in the Suf pathway, lack of detailed mechanistic descriptions prevents the rational design of small molecules to effectively disrupt the assembly process. During the previous funding period, regulation of the sulfur mobilization step was characterized in detail. The overall goal of this proposal is to investigate PPIs regulating cluster assembly and downstream trafficking. The hypothesis is that PPIs important for regulating cluster assembly and trafficking are governed by changes in the structure/dynamics of Suf proteins through the assembly process. We will test this hypothesis using a complementary, multi-pronged approach including hydrogen/deuterium exchange mass spectrometry (HDX-MS), protein crystallography, biophysical/biochemical characterization, and genetic complementation assays. To accomplish this broad approach, a research team with diverse backgrounds and a productive track-record has been assembled. The specific aims of this renewal proposal include: (1) characterization of Fe-S cluster-based regulation of sequential protein-protein interactions with SufBC2D, (2) determination of the role of ATP in the function of SufBC2D, and (3) identification of cluster trafficking interactions between the Suf system and the broader pool of Fe-S cluster carrier proteins. Completion of the experiments described in the proposal will provide a rigorous mechanistic description of how Fe-S cluster assembly and trafficking is regulated in the Suf pathway. These results can be leveraged to design potential antibiotics targeting bacterial Fe-S cluster pathways and may inspire novel therapeutic interventions for defects in analogous human pathways.

项目摘要 铁硫（FE-S）辅因子生物发生对于大多数活生物体至关重要。持续的长期目标 该更新建议是表征对细菌Fe-S簇至关重要的蛋白质 - 蛋白质相互作用（PPI） Suf（硫形成）途径的生物发生，该途径在原核生物中无处不在 病原体。由于细胞中游离铁和硫化物的毒性性质，Fe-S簇组装和运输高度很高 由复杂的蛋白质伙伴网络编排。在大肠杆菌中，Suf已经演变为 在氧化应激或铁饥饿条件下激活的紧急途径。在其他细菌中， 例如结核分枝杆菌和粪肠球菌，Suf途径是Fe-S的唯一来源 集群，使其成为抗生素发展的必要途径和潜在的靶标。 SUF途径包括 在六种蛋白质中，sufabcdse。 SUFS是一种半胱氨酸脱硫酶，可动员L-半胱氨酸的硫化硫化物（SO）。 Sufs产生的硫硫化物必须在交付给SUFB之前将其转移到转移硫酶蛋白SUFE，SUFE 在Sufbc2d群集脚手架上。在体内，Sufbc2d支架上的Fe-S群集组件取决于 SUFC的ATPase活性和铁的获得，这很可能受SUFD的调节。群集组装后， Sufbc2d将新生集群转移到Sufa进行下游贩运。而简单的功能分配 可用于SUF途径中的蛋白质，缺乏详细的机械描述可以阻止理性 小分子的设计以有效破坏组装过程。在上一个资金期间， 详细描述了硫动员步骤的调节。该提议的总体目标是 调查PPI调节集群组件和下游贩运的PPI。假设是PPI很重要 用于调节群集组装和运输，由SUF蛋白的结构/动力学变化控制 通过组装过程。我们将使用互补的多管齐下方法检验该假设 包括氢/氘交换质谱法（HDX-MS），蛋白质晶体学， 生物物理/生化特征和遗传互补测定法。实现这一广泛 Angred是一个具有不同背景和富有成效的田径记录的研究团队。这 该更新建议的具体目的包括：（1）基于Fe-S聚类的调节的表征 顺序蛋白质 - 蛋白质与Sufbc2d的相互作用，（2）确定ATP在功能中的作用 SUFBC2D和（3）识别SUF系统与更广泛的池之间的集群交互 Fe-S簇载体蛋白。提案中描述的实验的完成将提供严格的 Fe-S群集组装和运输方式在SUF途径中如何调节的机理描述。这些 可以利用结果来设计针对细菌Fe-S簇途径的潜在抗生素，并可能激发 针对类似人类途径缺陷的新型治疗干预措施。