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

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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.