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

智力优势：血红素是哺乳动物体内最丰富的循环铁来源。 因此，许多致病细菌，包括机会性铜绿假单胞菌，热切地追求其捕获和内化，以克服在其哺乳动物宿主中遇到的非常低的游离铁浓度，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病原菌部署了血红素获取系统（Has），其由分泌到细胞外空间的蛋白质（HasAp）和外膜受体（HasR）组成。 HasAp也被称为血载体，因为它有效地捕获血红蛋白-血红素并将其递送至受体用于随后的内化。 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，这种相互作用使HasAp能够从人类血红蛋白中“窃取”血红素。 特别是，研究人员试图获得结构，动态和机制的见解，以确定血红素从人类血红蛋白转移到HasAp的因素。 这一长期目标将通过实现三个主要目标来实现：1）阐明apo-HasAp的三维结构，2）鉴定当HasAp在血红素转移之前与血红蛋白结合时形成的遭遇复合物的结合界面，并破译HasAp结构元件的总体重组在分子识别和与血红蛋白结合中所起的作用，以及3）表征在血红素从血红蛋白转移到HasAp的早期阶段血红素-铁经历的配位和自旋状态的变化。 Moënne-Loccoz和里维拉实验室已联合起来，以满足拟议活动的跨学科需求。 里维拉实验室将进行NMR光谱研究，旨在解决没有血红素的HasAp的结构，绘制复合物的界面，并研究HasAp在相遇复合物中的动力学性质。Moënne-Loccoz实验室将进行快速混合淬灭实验，并结合UV-vis，EPR和共振拉曼，以描述血红蛋白血红素被HasAp捕获时的命运。 更广泛的影响：除了提供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中的低铁浓度的方式的分子见解之外，从这些研究中获得的基础知识可能为血红蛋白和HasAp之间相互作用的抑制剂的未来设计铺平道路。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和里维拉实验室的视频电话会议和年度访问将使学生接触到培养多学科研究所必需的智力多元化氛围。 这两个群体中存在的种族丰富的环境将为学生毕业后可能遇到的多方面工作环境做好准备。 该项目的合作精神将说明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在一个实验室中获得的结果;（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.