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: 8801246
• 负责人: Patrick Frantom
• 金额: $ 26.19万
• 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 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8801246
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; Affect; Allosteric Regulation; Antibiotic Resistance; Antibiotics; Architecture; Bacteria; Binding; Binding Sites; Biochemical Genetics; Biogenesis; Carrier Proteins; Catalysis; Chemistry; Complex; Coupled; Crystallography; Data; Deuterium; Development; Escherichia coli; Foundations; Genes; Goals; Host Defense; Hydrogen; Infection; Iron; Maps; Mass Spectrum Analysis; Mediating; Metals; Mycobacterium tuberculosis; NMR Spectroscopy; Nucleotides; Nutrient; Operon; Oxidative Stress; Pathogenesis; Pathway interactions; Play; Process; Protein Dynamics; Proteins; Public Health; Pyridoxal Phosphate; Reaction; Recombinant Proteins; Regulation; Role; Scaffolding Protein; Starvation; Stress; Structure; Sulfhydryl Compounds; Sulfur; System; Testing; Therapeutic; antibiotic design; base; cofactor; cysteine desulfurase; design; enzyme activity; genetic approach; in vivo; insight; iron metabolism; metalloenzyme; novel; pathogen; pathogenic bacteria; protein protein interaction; protein transport; public health relevance; reactive oxygen intermediate; research study; response; scaffold; trafficking

DESCRIPTION (provided by applicant): Many pathogenic bacteria must compete with the mammalian host for the essential metal iron, even as the host sequesters circulating iron and releases reactive oxygen intermediates to damage bacterial metalloenzymes as its first line of defense. Iron-sulfur (Fe-S) cluster cofactor biogenesis is critical to the survival of bacterial pathogens. A concerted multi-protein system encoded by the suf operon provides bacteria with a protected Fe-S biogenesis pathway under these conditions, which are detrimental to thiol and iron chemistry. The suf operon, conserved in Escherichia coli and in pathogenic Mycobacterium tuberculosis, encodes six proteins whose functions are still being elucidated. The SufS cysteine desulfurase requires the SufE sulfur transfer partner protein as well as the SufBC2D scaffold complex for full activity during sulfur trafficking. Sulfur donation from SufE to the SufB scaffold protein requires that SufB also be in a complex with SufC. The SufC ATPase is dependent on interactions with SufB and/or SufD for activation of the ATP hydrolysis cycle. In vivo Fe-S cluster assembly on SufB also requires the SufD protein. Finally, the SufA Fe-S cluster trafficking protein preferentially interacts with the [4Fe-4S] form of SufBC2D rather than the apo form of the scaffold complex. Thus Suf Fe-S cluster assembly is synchronized by a complicated network of protein- protein interactions (PPIs). The tight control of SufS and SufC enzyme activity and the careful regulation of sulfur and Fe-S cluster trafficking are critical for Suf funtion during iron starvation and oxidative stress in vivo. Our long-term goal is to characterize PPIs critical for iron metallocofactor biogenesis in order to disrupt those interactions with novel, rationally designed antibiotics. The hypothesis being tested in this proposal is that PPIs and their coupled conformational responses coordinate the steps of Suf stress-responsive Fe-S cluster biogenesis. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) will be combined with biophysical, biochemical, and genetic approaches to test this hypothesis. The specific aims of the proposal are to (1) define the full mechanism of sulfur transfer from SufS to SufE to SufB scaffold complexes, (2) characterize the conformational dynamics of the SufBC2D Fe-S cluster scaffold complex, and (3) determine the mechanism of Fe-S cluster transfer from SufB to the Fe-S carrier SufA. Completion of the experiments in this proposal will provide structural insight into the mechanistic roles of PPIs during Fe-S biogenesis by the Suf pathway. This insight will allow us to target key PPIs in the Suf pathway using novel antibiotic strategies.

描述（由申请人提供）：许多致病细菌必须与哺乳动物宿主竞争必需的金属铁，即使宿主隔离循环铁并释放了活性氧中间体，以损害细菌金属酶作为其第一条防御。铁硫（FE-S）簇辅因子生物发生对于细菌病原体的存活至关重要。由SUF操纵子编码的协同多蛋白系统在这些条件下为受保护的Fe-S生物发生途径提供了细菌，这对硫醇和铁化学有害。 SUF操纵子在大肠杆菌和致病性结核菌中保守的操纵子编码六种仍在阐明功能的蛋白质。 Sufs半胱氨酸脱硫酶需要Sufe Sulfur转移伴侣蛋白以及Sufbc2d支架复合物，以便在硫运输期间进行全部活性。从Sufe到Sufb支架的硫捐赠 蛋白质要求SUFB也与SUFC相结合。 SUFC ATPase取决于与SUFB和/或SUFD的相互作用，以激活ATP水解周期。 SUFB上的体内Fe-S簇组件也需要SUFD蛋白。最后，Sufa Fe-S聚类运输蛋白优先与SUFBC2D的[4FE-4S]形式相互作用，而不是支架复合物的Apo形式。因此，Suf Fe-S簇组装由复杂的蛋白质相互作用（PPI）网络同步。 SUFS和SUFC酶活性的严格控制以及硫和Fe-S簇运输的仔细调节对于在体内铁饥饿和氧化应激期间对于Suf造成的量至关重要。我们的长期目标是表征对铁金属生物生物发生至关重要的PPI，以破坏与新颖的，合理设计的抗生素的相互作用。在该提案中检验的假设是PPI及其耦合构象反应协调了SUF应激反应性FE-S簇生物发生的步骤。氢/氘交换质谱（HDX-MS）将与生物物理，生化和遗传方法结合使用，以检验该假设。该提案的具体目的是（1）定义从Sufs到Sufe到Sufb脚手架配合物的硫转移的完整机制，（2）表征Sufbc2d Fe-S-S Cluster支架复合物的构象动力学，（3）确定从SUFB转移到Fe-Scrier Sufa的机制。该提案中实验的完成将为SUF途径在Fe-S生物发生过程中PPI的机械作用提供结构性洞察力。这种见解将使我们能够使用新型的抗生素策略在SUF途径中靶向关键PPI。