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活性氧中间体以破坏细菌金属酶作为其第一道防线。铁硫（Fe-S）簇辅因子的生物发生对细菌病原体的生存至关重要。在这些条件下，由suf操纵子编码的协同多蛋白系统为细菌提供了一个受保护的Fe-S生物生成途径，这对硫醇和铁化学是有害的。在大肠杆菌和致病性结核分枝杆菌中保守的subf操纵子编码六种功能仍在阐明的蛋白质。SufS半胱氨酸脱硫酶需要SufE硫转移伙伴蛋白以及SufBC2D支架复合物才能在硫运输过程中充分发挥活性。从SufE到SufB支架的硫捐赠