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.

项目概要 铁硫 (Fe-S) 辅因子的生物发生对于大多数生物体至关重要。持续的长期目标 该更新提案旨在表征对细菌 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普遍存在，包括重要的 病原体。由于细胞中游离铁和硫化物的毒性，Fe-S簇的组装和运输高度依赖 由蛋白质伙伴的复杂网络精心设计。在大肠杆菌中，Suf 已进化为一种 在氧化应激或缺铁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 簇，使其成为抗生素开发的重要途径和潜在目标。 Suf 途径包括 六种蛋白质，SufABCDSE。 SufS 是一种半胱氨酸脱硫酶，可从 L-半胱氨酸中动员过硫化物 (So)。 SufS 产生的过硫化物必须先转移至转过硫酶蛋白 SufE，然后再输送至 SufB 在 SufBC2D 集群支架上。在体内，SufBC2D 支架上的 Fe-S 簇组装取决于 SufC 的 ATP 酶活性和铁的获取，可能受 SufD 调节。集群组装后， SufBC2D 将新生集群转移到 SufA 进行下游贩运。虽然简单的功能分配 虽然可用于 Suf 途径中的蛋白质，但缺乏详细的机制描述阻碍了合理的研究 设计小分子以有效破坏组装过程。在上一个资助期间， 详细描述了硫动员步骤的调节。该提案的总体目标是 研究调节集群组装和下游贩运的 PPI。假设 PPI 很重要 用于调节簇组装和运输的受 Suf 蛋白结构/动力学变化的控制 通过组装过程。我们将使用互补的多管齐下的方法来检验这个假设 包括氢/氘交换质谱 (HDX-MS)、蛋白质晶体学、 生物物理/生化表征和遗传互补测定。为了实现这一广泛 通过这种方法，我们组建了一支具有不同背景和卓有成效的研究团队。这 该更新提案的具体目标包括：(1) 基于 Fe-S 簇的调控的特征 与 SufBC2D 的连续蛋白质-蛋白质相互作用，(2) 确定 ATP 在功能中的作用 SufBC2D，以及 (3) 识别 Suf 系统与更广泛的群体之间的集群贩运相互作用 Fe-S簇载体蛋白。完成提案中描述的实验将提供严格的 Suf 途径中如何调节 Fe-S 簇组装和运输的机制描述。这些 结果可用于设计针对细菌 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治疗干预措施。