Conformational dynamics and allosteric regulation during stress-responsive metallocofactor assembly

项目总结 铁-硫(铁-S)辅因子的生物合成是大多数生物所必需的。持续的长期目标是 这一更新建议是为了表征对细菌Fe-S簇至关重要的蛋白质-蛋白质相互作用(PPI) 原核生物中普遍存在的硫磺形成途径的生物发生，包括重要的 病原体。由于细胞内游离铁和硫化物的毒性，铁-S簇的组装和运输能力很强 由一个复杂的蛋白质伙伴网络编排。在大肠杆菌中，SuF已进化为一种 在氧化应激或铁饥饿条件下被激活的紧急途径。在其他细菌中， 如结核分枝杆菌和粪肠球菌，SuF途径是铁-S的唯一来源 这使其成为抗生素开发的重要途径和潜在靶点。Suf途径包括 在六种蛋白质中，SufABCDSE。SuFS是一种半胱氨酸脱硫酶，能从L-半胱氨酸中动员过硫化物(SO)。 SufS产生的过硫化物必须转移到转硫酶蛋白Sufe，然后才能传递给SufB 在SufBC2D集群脚手架上。在体内，铁-S簇在SufBC2D支架上的组装依赖于 SuFC的ATPase活性和铁的获取，可能受SufD的调节。在集群组装之后， SufBC2D将新生的集群转移到Sufa进行下游贩运。虽然简单的函数赋值 是可用于Suf途径中的蛋白质的，缺乏详细的机制描述阻碍了理性的 设计的小分子可以有效地扰乱组装过程。在上一次筹资期间， 对硫磺动员步骤的调控进行了详细的表征。这项提案的总体目标是 调查规范集群组装和下游贩运的PPI。假设是PPI很重要 调节簇的组装和运输受Suf蛋白结构/动力学的变化控制 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一假设 包括氢/氢交换质谱仪(HDX-MS)、蛋白质结晶学、 生物物理/生化特征和遗传互补分析。要实现这一广泛的目标 在这方面，已经组建了一支具有不同背景和卓有成效的记录的研究小组。这个 这项更新建议的具体目的包括：(1)表征基于Fe-S集群的监管 蛋白质-蛋白质与SufBC2D的顺序相互作用，(2)ATP在细胞功能中作用的测定 SufBC2D，以及(3)确定Suf系统与更广泛的 铁-S簇状载体蛋白。完成提案中描述的实验将提供严格的 铁-S簇组装和运输如何在SuF途径中被调控的机制描述。这些 结果可用于设计针对细菌Fe-S簇途径的潜在抗生素，并可能启发 针对类似人类路径缺陷的新的治疗干预。

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.