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    The Structural Dynamics of Human Lipoxygenases Enzymatic and Regulatory Mechanisms

    Droege, Kristin Diane
    : https://etd.library.vanderbilt.edu/etd-08042017-000525
    http://hdl.handle.net/1803/13819
    : 2017-08-08

    Abstract

    Leukotrienes and lipoxins are lipid mediators of inflammation and their biosynthesis is associated with several chronic inflammatory and cardiovascular diseases. 5-Lipoxygenase (5-LOX) and 15-lipoxygenase (15-LOX) are key enzymes in the oxidation of arachidonic acid (AA) to form leukotrienes and lipoxins. Lipoxygenases are heavily regulated, non-heme dioxygenases. Their optimal activity is dependent on calcium binding to the PLAT domain and translocation from the cytosol to the membrane. 5-LOX also requires protein complex formation with 5-lipoxygenase activating protein (FLAP) for optimal activity in intact cells. Previous structure analysis has failed to identify conformation changes required for these regulatory mechanisms. We used hydrogen-deuterium (H/D) exchange mass spectrometry (MS) to investigate the structural dynamics of human 15-LOX-2 upon binding of Ca2+ and ligands, as well as upon membrane association. In addition, this work aimed to use H/D exchange to analyze protein and ligand interactions in the 5-LOX/FLAP complex. H/D exchange studies with 5-LOX and FLAP were limited due to 5-LOX loss of enzymatic activity over time points required for H/D exchange MS. However, a partial peptide map of FLAP revealed a putative AA binding site in the hydrophobic cavity formed by the FLAP homotrimer. Probing the solvent accessibility and backbone flexibility of 15-LOX-2 revealed high flexibility in the PLAT domain structure. Comparison of H/D exchange for soluble 15-LOX-2, soluble Ca2+ bound 15-LOX-2, and nanodisc-associated Ca2+ bound 15-LOX-2 indicates there are few differences in structural dynamics. Suggesting that changes in 15-LOX-2 structure are not required for membrane association. In addition, our results show that lipid binding to 15-LOX-2 active site in the catalytic domain induces changes in the structural dynamics in the PLAT domain and suggest a mechanism for allosteric regulation. Overall, our results challenge the previous hypothesis that Ca2+ induced structural changes of the 15-LOX-2 PLAT domain promote membrane association, and support the hypothesis that there is inter-domain communication in 15-LOX-2.
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