Collaborative Research: Molecular Mechanism of Hemoglobin-Heme Capture by the Hemophore (HasAp) Secreted by P. aeruginosa

智力优势：血红素是哺乳动物中最丰富的循环铁来源。因此，包括机会性铜绿假单胞菌在内的许多致病菌，为了克服在哺乳动物宿主中遇到的极低的游离铁浓度，急切地追求捕获和内化，这并不奇怪。为了捕获血红素，包括铜绿假单胞菌在内的几种致病菌部署了一个血红素获取系统（Has），该系统由分泌到细胞外空间的蛋白质（HasAp）和外膜受体（HasR）组成。HasAp也被称为血细胞，因为它能有效地捕获血红素并将其传递给受体，以便随后内化。该项目旨在实现对蛋白质-蛋白质相互作用的基本分子水平的理解，这种相互作用允许HasAp从人类血红蛋白中“窃取”血红素。特别是，研究人员试图获得决定血红素从人血红蛋白向HasAp转移的因素的结构、动态和机制方面的见解。这一长期目标将通过追求三个主要目标来实现：1)阐明apo-HasAp的三维结构；2)确定在血红素转移之前，HasAp与血红蛋白结合时形成的偶遇复合物的结合界面，破译HasAp结构元件的总体重组在血红蛋白分子识别和结合中所起的作用；3)表征血红素从血红蛋白转移到HasAp的早期血红素铁的配位和自旋状态的变化。Moënne-Loccoz和Rivera实验室联合起来，以满足拟议活动的跨学科需求。Rivera实验室将进行核磁共振波谱研究，旨在解决缺乏血红素的HasAp结构，绘制络合物的界面，并研究HasAp在遇到络合物中的动力学特性。Moënne-Loccoz实验室将进行快速混合猝灭实验，结合UV-vis， EPR和共振拉曼，以描述血红蛋白血红素在被HasAp捕获时的命运。更广泛的影响：除了为铜绿假单胞菌从血红蛋白中捕获血红素以克服哺乳动物宿主体内低铁浓度的方式提供分子洞察力外，这些研究获得的基础知识可能为未来设计血红蛋白与HasAp相互作用抑制剂铺平道路。该项目的多学科性质将为所有学术水平的学生提供机会。来自Moënne-Loccoz和Rivera实验室的视频电话会议和年度访问将使学生们接触到培养多学科研究所必需的智力多样化的氛围。在这两个群体中存在的丰富的民族环境将使学生为毕业后可能遇到的多方面的工作环境做好准备。这个项目的合作精神将说明一个基础广泛的方法的好处：(1)解决复杂的问题；(2)在全球范围内有效地解释一个实验室获得的结果；从而(3)最大限度地提高这项研究对更大的科学界和公众的影响。

Intellectual Merit: Heme is the most abundant source of circulating iron in mammals. It is therefore not surprising that many pathogenic bacteria, including the opportunistic Pseudomonas aeruginosa, avidly pursue its capture and internalization in order to overcome the very low free-iron concentrations encountered in their mammalian hosts. To capture heme, several pathogenic bacteria, including P. aeruginosa, deploy a heme acquisition system (Has), which consists of a protein secreted to the extracellular space (HasAp) and an outer membrane receptor (HasR). HasAp is also termed a hemophore because it efficiently captures hemoglobin-heme and delivers it to the receptor for subsequent internalization. The project aims to achieve fundamental molecular level understanding of the protein-protein interactions that allow HasAp to "steal" heme from human hemoglobin. In particular, the investigators seek to gain structural, dynamic and mechanistic insights into the factors that determine the transfer of heme from human hemoglobin to HasAp. This long-range goal will be reached by pursuing three main objectives: 1) Elucidate the three dimensional structure of apo-HasAp, 2) Identify the binding interface of the encounter complex that forms when HasAp binds to hemoglobin, prior to heme transfer, and decipher the role played by the gross reorganization of HasAp structural elements in the molecular recognition and binding to hemoglobin, and 3) Characterize the changes in coordination and spin state experienced by the heme-iron in the early stages of heme transfer from hemoglobin to HasAp. The Moënne-Loccoz and Rivera laboratories have joined forces to meet the interdisciplinary demands of the proposed activities. The Rivera lab will conduct the NMR spectroscopic studies aimed at solving the structure of HasAp devoid of heme, map the interface of the complex and study the dynamical properties of HasAp in the encounter complex. The Moënne-Loccoz lab will carry out the rapid-mix-quench experiments coupled to UV-vis, EPR and resonance Raman necessary to delineate the fate of the hemoglobin-heme as it is captured by HasAp. Broader Impacts: In addition to providing molecular insight into the manner by which P. aeruginosa captures heme from hemoglobin to overcome the low iron concentrations in a mammalian host, the fundamental knowledge derived from these investigations may pave the way for the future design of inhibitors to the interactions between hemoglobin and HasAp. The multidisciplinary nature of the project will provide opportunities for students at all academic levels. Video-conference calls and yearly visits from the Moënne-Loccoz and Rivera labs will expose the students to the intellectually diverse atmosphere that is necessary to nurture multidisciplinary research. The ethnically rich environment present in both groups will prepare students for the multifaceted work environment they are likely to encounter after graduation. The collaborative spirit of this project will illustrate the benefits of a broad-based approach for (1) solving complex problems, (2) effectively interpreting results obtained in one laboratory within a global context; and thus (3) maximizing the impact on this research on the greater scientific community and the general public.