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.
Antibiotic resistance is a major public concern as some of the first-line antibiotics are decades old, and resistance to them becomes widespread. Most currently used antibiotics target similar biological processes such as oligonucleotides, cell membrane, or protein synthesis. To counteract ever-evolving resistance, it is important to find new bacterial vulnerabilities and develop new targets. In this context, it is noteworthy that all studied pathogens require iron. Given that approximately 75% of the total iron in humans is bound by heme and incorporated into hemoglobin, the latter constitutes a pathogen main source of iron. P. aeruginosa has become a prevalent cause of hospital infections, and is the primary cause of chronic lung infections in individuals with cystic fibrosis. P. aeruginosa uses the heme acquisition system (has) to "steal" heme from its host hemoglobin, in order to obtain iron. Has consists of a heme receptor (HasR), which is located on the bacterial outer membrane, and HasAp, a protein secreted by P. aeruginosa, which is thought to "steal" heme from hemoglobin and deliver it to HasR, for bacterial uptake and subsequent utilization of heme-iron. The high affinity of HasA-like molecules for heme has given rise to the term hemophore. In this collaborative research the Rivera and Moënne-Loccoz laboratories have joined forces to understand the mechanism by which HasAp binds heme, with the long-term objective of targeting HasAp for possible antibiotic development. X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and fast spectroscopic methods (UV-vis, resonance Raman and electron paramagnetic resonance) were utilized to study the manner in which heme binds to apo-HasAp. It was found that in the structure of apo-HasAp (devoid of heme) a loop offers a hydrophobic platform where heme can "land" and then coordinate Tyr75. A second loop, which in the apo-HasAp structure looks like an "open lid", detects the presence of heme in the platform below and initiates a "closing process" which culminates in coordination of the top face of the heme by His32 and the trapping of heme. It was also determined that the initial binding of heme to the hydrophobic platform takes place in a few milliseconds. Closing of the lid, on the other hand, is a significantly slower process, which is completed in a timescale of seconds. These new insights suggest that the hydrophobic platform where heme lands is very important for heme capture by hemophores, and we speculate that the opening and closing of the lid may be related to protein-protein interactions that enable heme capture or heme delivery to the receptor HasR. Interestingly, our latest discovery shows that the "open lid" observed in hemophores from the pathogens Pseudomonas aeruginosa (HasAp) and Serratia marcescens (HasAs) is not conserved in a hemophore from Yersinia pestis (HasAyp). In the latter, the hemophore captures heme with minimal reorganization of protein structure, and binds heme only with Tyr75, unlike HasAp and HasAs, which bind heme with Tyr75 and His32. These interesting differences provide a unique handle for future studies directed at understanding the roles played by the two loops in hemophores in directing protein-protein interactions and heme delivery to the receptor HasR. Details of our findings have been presented in publications (1-5). Publications: 1. Alontaga, A. Y., Rodríguez, J. C., Schönbrunn, E., Becker, A., Funke, T., Yukl, E. T., Hayashi, T., Stobaugh, J., Moënne-Loccoz, P., and Rivera, M. (2009) Structural Characterization of the Hemophore HasAp from Pseudomonas aeruginosa: NMR Spectroscopy Reveals Protein-Protein Interactions Between Holo-HasAp and Hemoglobin, Biochemistry 48, 96-109. 2. Jepkorir, G., Rodríguez, J. C., Rui, H., Im, W., Lovell, S., Battaille, K. P., Alontaga, A. Y., Yukl, E. T., Moënne-Loccoz, P., and Rivera, M. (2010) Structural, NMR Spectroscopic and Computational Investigation of Hemin Loading in the Hemophore HasAp from Pseudomonas aeruginosa, J. Am. Chem. Soc. 132, 9857-9872. 3. Yukl, E. T., Jepkorir, G., Alontaga, A., Pautsch, L., Rodriguez, J. C., Rivera, M., and Moënne-Loccoz, P. (2010) Kinetic and Spectroscopic Studies of Hemin Acquisition in the Hemophore HasAp from Pseudomonas aeruginosa, Biochemistry 49, 6646-6654. 4. Kumar, R., Lovell, S., Matsumura, H., Battaile, K. P., Moenne-Loccoz, P., and Rivera, M. (2013) The Hemophore HasA from Yersinia pestis (HasAyp) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change, Biochemistry 52, 2705-2707. 5. Benson, D. R., and Rivera, M. (2013) Heme Uptake and Metabolism in Bacteria, Met. Ions Life Sci. 12, 279-332.
