Satellite Program Oral The 48th Lorne Conference on Protein Structure and Function 2023

Cryo-EM structures of human arachidonate 12S-Lipoxygenase (12-LOX) bound to endogenous and exogenous inhibitors (#36)

Jesse I Mobbs 1 2 , Katrina A Black 2 3 4 , Michelle Tran 5 , Hari Hari Venugopal 6 , Theodore Holman 5 , Michael Holinstat 7 8 , David M Thal 1 2 , Alisa Glukhova 1 2 3 4 9
  1. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
  2. ARC Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia
  3. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  4. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  5. Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California, USA
  6. Ramaciotti Centre for Cryo-Electron Microscopy , Monash University, Clayton, Victoria, Australia
  7. Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA
  8. Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA
  9. Department of Biochemistry and Pharmacology, The University of Melbourne, Melbourne, Victoria, Australia

Arachidonate 12-lipoxygenase (12-LOX) is one of the primary enzymes involved in platelet activation. 12-LOX oxidises arachidonic acid to produce 12-hydroperoxyeicosatetraeonic acid (12S-HpETE), a small molecule with a pro-thrombotic role in thrombogenesis. Because of this, 12-LOX dysregulation leads to occlusive thrombotic events, myocardial infarction, and stroke. 12-LOX is therefore considered a prime drug candidate for treating ischemic heart disease and stroke. Indeed, the highly selective 12-LOX inhibitor, ML355, has passed a phase I clinical trial to treat heparin-induced thrombocytopenia and has received fast-track designation by the FDA. Despite its promising roles in antiplatelet therapies, the exact mechanisms of how 12-LOX affects platelet activation remain unclear, and the lack of structural information limits further drug discovery of this enzyme.

Here we have used single-particle cryo-EM to determine the first human structures of 12-LOX. These structures observed 12-LOX to exist in multiple oligomeric states, from monomer to hexamer, that likely play a role in its catalytic activity and membrane association. Furthermore, 12-LOX was captured in multiple conformational states, “open” and “closed”, likely representing different time points in the catalytic cycle. Lastly, due to the high resolution of these structures, we identified an endogenous 12-LOX inhibitor, a long-chain acyl-CoA, bound in the catalytic site of the 12-LOX enzyme. In addition, we identified ML355 binding to a putative allosteric binding site in the hexameric oligomer. These high-resolution structures of 12-LOX provide unique insights into the assembly of 12-LOX oligomers, provide an understanding of its catalytic mechanism, and pave the way for further drug development of the 12-LOX enzyme.