Pass the 12-LOX!

In this issue of Blood, Mobbs et al¹ report high-resolution structures of the enzyme 12S-lipoxygenase (12-LOX) obtained by cryogenic electron microscopy. The study elucidates the oligomeric states, conformational plasticity, and binding interactions of an intriguing enzyme involved in platelet activity and thrombosis. The new knowledge may boost the development of structure-based inhibitors.

The enzyme 12-LOX is expressed in platelets, where it promotes activation of αIIbβ3, glycoprotein VI, and protease activated receptor (PAR4), as well as in human islets, epidermal keratinocytes, and several tumor cell lines.² For this reason, 12-LOX constitutes an important molecular target for the treatment of thrombosis, heparin-induced thrombocytopenia (HIT), and platelet-mediated cancer progression. Many aspects of the function of 12-LOX in platelet biology and signaling remain incompletely understood. There is insufficient structural information and a lack of selective inhibitors to be used in animal models, where results from knockout mice have been conflicting.^3,4 As for other proteins relevant to hemostasis and thrombosis, advances in the structural biology of 12-LOX would afford a better understanding of function and eventually guide the development of selective inhibitors for therapeutic applications.

In this issue of Blood, Mobbs et al take advantage of the unique features of cryogenic electron microscopy (cryo-EM) in solving the structure of macromolecules under nativelike conditions. The study captures the molecular plasticity of 12-LOX for the first time from structures solved at very high resolution (1.7-2.8 Å) and from particles imaged from the same cryogenic grid. A snapshot of 12-LOX emerges with the protein distributed among different oligomeric forms and with the active site assuming “open” or “closed” conformations. The results are highly significant and will impact future functional and translational studies of this intriguing enzyme.

The structural organization of 12-LOX recapitulates the fold of typical LOX enzymes, with the active site housing a catalytic nonheme Fe²⁺ atom bound to 3 conserved His residues.⁵ The entrance to the active site is lined by an arched helix and an α2 helix in an extended conformation. Protein from a “dimer peak” obtained from size exclusion chromatography produces multiple high-resolution cryo-EM structures of monomers, dimers, tetramers, and hexamers of 12-LOX from the same cryogenic grid. The dimer provides the functional unit of 12-LOX, with individual monomers assembled “head-to-toe” and stabilized by Van der Waals interactions and numerous H-bonds. The arrangement confirms the results of previous small-angle X-ray scattering (SAXS) analysis.⁶ New for the cryo-EM study is the elucidation of higher oligomers of 12-LOX that show how the arrangement of the dimer is retained when tetramers and hexamers are assembled as dimers and trimers of dimers, respectively. The 2-ring arrangement of the hexamer creates a pore with a diameter of ∼30 Å, which may have a physiological role and should stimulate further investigation. The hexamer is stabilized by a disulfide bond, unlike the dimer and tetramer, which suggests that formation of higher oligomers of 12-LOX may be sensitive to the oxidative environment of the cell. The different oligomeric states of 12-LOX likely regulate enzyme activity and interaction with membranes. The active site entrance and membrane-binding residues are positioned on the same surface in the 12-LOX dimer but are covered by dimer-dimer contacts in the tetramer and hexamer. Therefore, the dimer may be the only active oligomer of 12-LOX. Notably, the individual subunits of the dimer are not identical in conformation. A shift of the α2 helix keeps the active site “open” in one subunit but “closed” in the other. This feature of 12-LOX may impact ligand binding and catalysis, especially if the 2 conformations of the active site preexist in equilibrium and interconvert on a time scale comparable to substrate binding, as observed in allosteric enzymes.⁷ 

To further support the functional importance of different oligomeric states of 12-LOX, a fatty acid acyl-coenzyme A (CoA) molecule of unconfirmed nature is found in the active site in the open form of all 12-LOX oligomers, but resolved with confidence only in the tetramer and hexamer. The observation is physiologically relevant because fatty acid acyl-CoA derivatives inhibit platelet aggregation in a chain-, length-, and saturation-dependent manner through interference with P2Y1 and P2Y12 receptors.⁸ It is possible that fatty acid acyl-CoA, especially oleyl-CoA identified by the authors from enzymatic assays, functions as endogenous inhibitor of 12-LOX and contributes to suppress platelet aggregation. Related to this issue is new structural information on the binding mode of ML355, a selective and potent inhibitor of human platelet 12-LOX⁹ capable of preventing thrombus formation without impairing hemostasis.¹⁰ The inhibitor recently concluded phase I clinical trials for the treatment of HIT and received fast-track designation by the Food and Drug Administration. It is conceivable that ML355 may also find applications as an anticancer and antimetastatic agent. Previous docking and mutagenesis studies predicted that ML355 would penetrate the active site of 12-LOX, but the cryo-EM structure instead shows ML355 binding to an allosteric site at the entrance to the active site and only in the hexamer. The very high resolution of the cryo-EM structure also enables direct validation of detailed contacts responsible for formation of the complex by site-directed mutagenesis. The stage is now set for further optimization of ML355 and the development of new inhibitors of 12-LOX using structure-based approaches.

The work by Mobbs et al¹ broadens our understanding of 12-LOX at the structural level and will definitely impact future studies of this intriguing enzyme as a target of antiplatelet and possibly anticancer therapy. New efforts should address the functional properties of each oligomeric state of 12-LOX, the role of the open and closed states of the active site in catalysis, and the biological consequences of inhibition by endogenous fatty acid acyl-CoA. Importantly, the structure of 12-LOX pushes the limits of resolution that can be achieved with cryo-EM when investigating proteins relevant to hemostasis and thrombosis. It sets a high bar for future studies, but also shows what is possible with application of this revolutionary technique. So, pass the 12-LOX!

Conflict-of-interest disclosure: The author declares no competing financial interests.