Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors

Abstract

Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which 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 high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, 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 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.

Graphical abstract

Figure 1.

Different oligomeric forms of 12-LOX. (A) SEC UV absorbance trace (280 nm ) of 12-LOX purification. 12-LOX separated as 2 distinct peaks, 1 corresponding to a tetramer (red box) and the other corresponding to a dimer (blue box). (B) Cryo-EM map of a 12-LOX tetramer from tetrameric SEC peak. (C) Cryo-EM maps of different 12-LOX oligomers resolved from dimeric SEC peak.

Figure 2.

Oligomeric structures of 12-LOX. Models of 12-LOX as (A) monomer, (B) dimer, (C) tetramer, and (D) hexamer. Each subunit is represented by a different color and the α2-helix colored in pink and an arched helix in cyan. The graphical representation of each oligomeric state at the bottom left and inset details the oligomeric interface. Interacting amino acids are shown as sticks. (D) Cys89 (red) contributes a disulfide bridge to the interface of the hexamer. The Fe atom is shown as a red sphere.

Figure 3.

Conformational changes in the 12-LOX dimer. (A) Surface representation of 12-LOX in the open (left) and closed states (right), showing the active site cavity of the dimer 12-LOX subunits. In the open conformation, the cavity is occupied by a small molecule. (B) An alignment of open and closed states shows a 23° rotation and unwinding of the N-terminal residues of the α2-helix. The inset shows the zoomed-in view of the active site entrance. The α2-helix is in cyan.

Figure 4.

Acyl-CoA binding site in the 12-LOX tetramer. (A) Model of a 12-LOX tetramer, with density in the catalytic site shown in cyan. The graphical representation is shown in the right corner. (B) Acyl-CoA model and the density. Density is shown as a transparent surface, and the model as sticks colored by the heteroatoms. (C) Model of acyl-CoA within the catalytic site of 12-LOX. (D-E) 12-LOX residues in contact with acyl-CoA (orange) are shown as sticks. (D) Interactions of the adenosine triphosphate group. (E) Interactions of the acyl tail. (F) Conformational changes in the residues in contact with acyl-CoA. Acyl-CoA--bound subunit is shown in purple and unbound is shown in pink. Iron atom is shown as a red sphere.

Figure 5.

ML355 and AA binding sites in the 12-LOX hexamer. (A) Model of the 12-LOX hexamer with density at the catalytic site shown in gray. The graphical representation is shown in the bottom left corner. (B) Density of AA and ML355. Density is shown as a transparent surface, and the models as sticks (orange for ML355 and pink for AA) colored by heteroatoms. (C) Model of 12-LOX bound to ML355 and AA. (D-E) 12-LOX residues in contact with (D) ML355 (orange) and (E) AA (pink) are shown as sticks. Iron atom is shown as a red sphere.