The Structural Dynamics of Human Lipoxygenases Enzymatic and Regulatory Mechanisms

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.