Conformational dynamics and allosteric regulation during stress-responsive metallocofactor assembly

描述（由申请人提供）：许多病原菌必须与哺乳动物宿主竞争必需的金属铁，即使宿主螯合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。铁硫簇辅因子的生物合成对细菌病原体的生存至关重要。由suf操纵子编码的协同多蛋白系统在这些条件下为细菌提供了受保护的Fe-S生物合成途径，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suf操纵子编码六种蛋白质，其功能仍在阐明中。SufS半胱氨酸脱硫酶需要SufE硫转移伴侣蛋白以及SufBC 2D支架复合物以在硫运输期间具有完全活性。从SufE到SufB支架的硫供给 蛋白质需要SufB也与SufC形成复合物。SufC ATP酶依赖于与SufB和/或SufD的相互作用来激活ATP水解循环。体内Fe-S簇在SufB上的组装也需要SufD蛋白。最后，SufA Fe-S簇运输蛋白优先与[4Fe-4S]形式的SufBC 2D而不是载脂蛋白形式的支架复合物相互作用。因此，Suf Fe-S簇组装通过蛋白质-蛋白质相互作用（PPI）的复杂网络同步。SufS和SufC酶活性的严格控制以及硫和Fe-S簇运输的仔细调节是体内铁饥饿和氧化应激过程中Suf功能的关键。我们的长期目标是表征对铁金属辅因子生物合成至关重要的PPI，以破坏与新型，合理设计的抗生素的相互作用。在这个建议中测试的假设是PPI和它们的耦合构象响应协调的步骤Suf应力响应的Fe-S簇生物发生。氢/氘交换质谱（HDX-MS）将与生物物理，生物化学和遗传学方法相结合，以测试这一假设。该提案的具体目的是（1）定义硫从SufS转移到SufE再到SufB支架复合物的完整机制，（2）表征SufBC 2D Fe-S簇支架复合物的构象动力学，以及（3）确定Fe-S簇从SufB转移到Fe-S载体SufA的机制。完成本提案中的实验将提供结构洞察PPIs在Fe-S生物合成过程中通过Suf途径的机制作用。这一见解将使我们能够使用新的抗生素策略靶向Suf途径中的关键PPI。

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.