Project Summary/Abstract Lipoxygenases are enzymes that contribute to the initiation and resolution of inflammation by generating specific lipid oxygenation products. These proteins rest in the cytosol and target to the membrane upon Ca2+ stimulation where substrate is acquired. The objective of this application is to revel how the conformational state regulates membrane targeting and substrate acquisition for two human lipoxygenase enzymes. The long- term goal is to elucidate the full molecular details of these human lipoxygenases in conformations bound to lipid substrate and small molecule inhibitors to aid in structure-based drug discovery. The rationale for the proposed research is enzymes that bind to the membrane transiently should adopt distinct conformations for differentiation between the two subcellular locations of the cytoplasm and the membrane by burial or exposure of hydrophobic residues. The objective of this project will be accomplished by two specific aims: (1) Elucidating the catalytically active and open form of human 5-lipoxygenase. Previous structures of human 5-lipoxygenase have been determined in a “closed” form with the active site inaccessible or in an incomplete “open” form with key peptide regions that define the active site unresolved by X-ray crystallography. Our research approach combines the design of strategic site-directed mutants to unlock and promote an “open” conformation that will be validated by kinetics and Hydroden Deuterium eXchange by Mass Spectrometry (HDX-MS). High-resolution structures of variants of 5-lipoxygenase that favor an “opened” form will be aggressively pursued. (2) The role of conformational flexibility in substrate acquisition at the membrane by 15-lipoxygenase-2. Lipid-analog detergents appear to be necessary for crystallization of 15-lipoxygenase-2. We will confirm lipid-binding sites on 15-LOX-2 through HDX-MS and X-ray crystallography. X-ray reflectivity and grazing incidence X-ray diffraction of 15-lipoxygenase-2 bound to a monolayer in a Langmuir trough will provide the most native structural data of a lipoxygenase at the membrane. The proposed project is significant because it will delineate the role lipids have on the conformational remodeling of a peripheral membrane-binding protein. We will reveal the molecular determinants that are responsible for substrate acquisition of two human lipoxygenases by (a) the design of new variants of human LOXs that bind substrate with altered kinetic constants, (b) HDX-MS of the enzymes in the presence of lipid-analog detergents, and (c) solve the structure of the enzymes bound to lipid-analog detergents and substrate. This study is innovative because it (a) develops new variants of human 5-lipoxygenase with strategic mutations to promote an “opened” form, (b) combines the methods of HDX-MS to provide rationale feedback of conformational flexibility to our X-ray crystallography efforts, and (c) will reveal the orientation and the depth of interact...