Conformational dynamics and allosteric regulation during stress-responsive metallocofactor assembly

项目摘要 铁硫（Fe-S）辅因子是大多数生物体的必需物质。持续的长期目标 这一更新建议是表征细菌Fe-S簇关键的蛋白质-蛋白质相互作用（PPI） 通过Suf（硫形成）途径的生物发生，这在原核生物中普遍存在，包括重要的 病原体由于游离铁和硫化物在细胞中的毒性，Fe-S簇组装和运输是高度不稳定的。 由一个复杂的蛋白质伙伴网络编排。在大肠杆菌中，Suf已经进化为充当 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， 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靶点。Suf途径包括 六种蛋白质，SufABCDSE。SufS是半胱氨酸脱硫酶，其从L-半胱氨酸动员过硫化物（So）。 由SufS产生的过硫化物必须在传递到SufB之前转移到转过硫酸酶蛋白SufE 在SufBC 2D簇支架上。在体内，SufBC 2D支架上的Fe-S簇组装依赖于 SufC的ATP酶活性和铁的获得，这可能是由SufD调节。在集群组装之后， SufBC 2D将新生簇转移到SufA进行下游贩运。虽然简单的功能分配 可用于Suf途径中的蛋白质，缺乏详细的机制描述阻止了合理的 小分子的设计，以有效地破坏组装过程。在上一个供资期间， 详细表征了硫活化步骤的调节。本提案的总体目标是 研究PPI调节簇组装和下游贩运。假设PPI重要 调节簇组装和运输的机制是由Suf蛋白的结构/动力学变化决定的 通过装配过程。我们将使用一种互补的、多管齐下的方法来检验这一假设 包括氢/氘交换质谱（HDX-MS），蛋白质晶体学， 生物物理/生物化学表征和遗传互补测定。为了实现这一广泛的 在这种方法下，组建了一个具有不同背景和富有成效的跟踪记录的研究团队。的 该更新建议的具体目标包括：（1）表征基于Fe-S簇的调节， （2）确定ATP在SufBC 2D功能中的作用， SufBC 2D，以及（3）识别Suf系统与更广泛的 Fe-S簇载体蛋白。完成提案中描述的实验将提供一个严格的 Fe-S簇组装和运输如何在Suf途径中调节的机制描述。这些 结果可以用来设计潜在的抗生素靶向细菌Fe-S簇途径，并可能启发 针对类似人类途径缺陷的新型治疗干预。

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.