The Mechanism of Fe2+ Transport by the Bacterial Ferrous Iron Uptake System Feo

说明(申请人提供)：铁是许多生命过程所必需的微量营养素，因此是病原菌增殖的限速生长因子。与Fe3+获取系统不同，在理解原核生物如何积累Fe2+方面存在着根本的差距。在缺氧条件下，例如在GI轨道上，Fe2+比Fe3+更丰富，相对未知的亚铁摄取系统FeO负责Fe2+的获取。最重要的FeO组分是FeoB，它是一种跨膜蛋白，含有与真核G蛋白偶联受体和信号蛋白相似的N-末端GTP结合和鸟嘌呤解离抑制因子(GDI)结构域。因此，FeoB被推测为人类G蛋白的祖先前体。由于敲除FeoB细菌株难以在小鼠模型的胃肠道定植，FeoB代表了一个有吸引力的抗菌靶点。本项目的最终目的是阐明FeO促进原核生物获取Fe2+的分子机制。目前，FeO系统如何促进Fe2+的转运尚不清楚。它被认为既可以作为GTP门控离子通道，也可以作为GTP供能的活性转运蛋白，或者作为GTP调节的信号和Fe2+敏感蛋白。为了在这些机制中辨别，将追求四个具体目标。首先，将对FeoB GTP酶活性的动力学进行表征。然后检测Fe2+和第二种FeO蛋白FeoA对FeoB GTP酶活性的影响。在第一个目标下，将通过研究FeoA-FeoB的相互作用来进一步定义FeoA的功能。其次，为了区分所提出的信号和转运机制，将使用含FeoB的膜小泡进行Fe2+摄取分析。这些相同的检测方法将用于评估FeoB的金属离子特异性。第三，序列分析表明，几个保守的跨膜残基是金属离子选择性和/或跨膜区和细胞质区域之间通讯的关键。将进行诱变研究，以探索GTP结合和Fe2+转运是如何耦合的。最后，将确定胞质FeoB结构域的apo、GTP和GDP结合形式的晶体结构，以提供对GTP酶活性和FeoB调控的机械性见解。综上所述，这些实验将阐明在原核生物中广泛使用的一种新的金属运输系统的机制，这对它们的生存是独一无二的。此外，拟议的研究有望促进对金属离子吸收和调节的理解。 与公共健康相关：这项建议将研究FeO系统促进原核生物在缺氧或还原条件下积累Fe2+的分子机制，例如在胃肠道中发现的那些条件。由于FeO系统是原核生物所特有的，对它们的生长至关重要，了解FeO控制Fe2+摄取过程的机制将有助于未来开发针对病原体的靶向治疗策略。

DESCRIPTION (provided by applicant): Iron is an essential micronutrient required for numerous life processes and therefore a rate-limiting growth factor for the proliferation of pathogenic bacteria. As opposed to Fe3+ acquisition systems, there is a fundamental gap in understanding how prokaryotes accumulate Fe2+. Under anoxic conditions, such as that of the GI track where Fe2+ is more abundant than Fe3+, the relatively uncharacterized ferrous iron uptake system, Feo, is responsible for Fe2+ acquisition. The most essential Feo component is FeoB, a transmembrane protein that contains N-terminal GTP-binding and guanine dissociation inhibitor (GDI) domains similar to those found in eukaryotic G-protein coupled receptors and signaling proteins. For this reason, FeoB is speculated to be an ancestral precursor to human G-proteins. Because knockout FeoB bacteria strains have difficulty colonizing GI tracts of mouse models, FeoB represents an attractive antimicrobial target. The ultimate goal of this project is to elucidate the molecular mechanism by which Feo facilities Fe2+ acquisition by prokaryotes. Presently, it is unknown how the Feo system acts to facilitate Fe2+ transport. It is postulated to act either as a GTP-gated ion channel, a GTP-energized active transporter, or a GTP-regulated signaling and Fe2+-sensing protein. To discern among these mechanisms, four specific aims will be pursued. First, the kinetics of the FeoB GTPase activity will be characterized. Then the influence of Fe2+ and a second Feo protein, FeoA, on the FeoB GTPase activity tested. Under the first aim, the function of FeoA will be further defined by examining FeoA- FeoB interactions. Second, in order to distinguish between the proposed signaling and transport mechanisms, Fe2+ uptake assays using FeoB-containing membrane vesicles will be conducted. These same assays will be used to assess the metal ion specificity of FeoB. Third, sequence analysis suggests several conserved transmembrane residues are key for metal ion selectivity and/or communication between the transmembrane and cytosolic regions. Mutagenic studies will be conducted to probe how GTP binding and Fe2+ transport are coupled. Lastly, crystal structures of the cytosolic FeoB domain in its apo, GTP and GDP bound forms will be determined to provide mechanistic insight into GTPase activity and the regulation of FeoB. Taken together, these experiments will shed light on the mechanism of a novel metal transport system used broadly among prokaryotes that is unique to their survival. In addition, the proposed research is expected to advance understanding of metal ion uptake and regulation. PUBLIC HEALTH RELEVANCE: This proposal will examine the molecular mechanisms by which the Feo system facilitates Fe2+ accumulation by prokaryotes under anoxic or reducing conditions, such as those found in the GI tract. Because the Feo system is unique to prokaryotes and vital for their growth, understanding the mechanism that governs Fe2+ uptake processes by Feo will allow for the future development of targeted therapeutic strategies against pathogens.