Abstract
Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation and regulation of its activity has been targeted for treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms of how it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine the high-resolution structures (1.7 Å - 2.8 Å) of human 12-LOX for the first time. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may impact its catalytic activity and membrane association. We also identified different conformations within a 12-LOX dimer, likely representing different time points in its catalytic cycle. Furthermore, we were able to identify small molecules bound to the 12-LOX structures. The active site of the 12-LOX tetramer is occupied by an endogenous 12-LOX inhibitor, a long-chain acyl-Coenzyme A. Additionally, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase I clinical trial for treating heparin-induced thrombocytopenia and has received fast-track designation by the FDA. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, its catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.
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