The structure of human 15-lipoxygenase-2 with a substrate mimic

Abstract

Atherosclerosis is associated with chronic inflammation occurring over decades. The enzyme 15-lipoxygenase-2 (15-LOX-2) is highly expressed in large atherosclerotic plaques, and its activity has been linked to the progression of macrophages to the lipid-laden foam cells present in atherosclerotic plaques. We report here the crystal structure of human 15-LOX-2 in complex with an inhibitor that appears to bind as a substrate mimic. 15-LOX-2 contains a long loop, composed of hydrophobic amino acids, which projects from the amino-terminal membrane-binding domain. The loop is flanked by two Ca(2+)-binding sites that confer Ca(2+)-dependent membrane binding. A comparison of the human 15-LOX-2 and 5-LOX structures reveals similarities at the active sites, as well striking differences that can be exploited for design of isoform-selective inhibitors.

Keywords: Atherosclerosis; Eicosanoid-specific Enzymes; Fatty Acid Oxidation; Lipoxygenase Pathway; Protein Structure.

FIGURE 1.

Overlay of 15-LOX-2 and stable 5-LOX. A, schematic renderings of 15-LOX-2 (green) and stable 5-LOX (pink). Ca²⁺ is shown as blue spheres, and the catalytic iron is in rust. B, detail of the difference in positioning of helix α2. Side chains from the 5-LOX segment occupy the shallower half of the 15-LOX-2 inhibitor binding site.

FIGURE 2.

15-LOX-2 and membrane binding. A, surface rendering of 15-LOX-2. The putative membrane insertion loop projects from the amino-terminal PLAT domain; prolines present in the loop are colored in red. B, rotation 90° about the horizontal, looking into the active site (C8E4, teal). The Ca²⁺-binding sites (blue spheres) are on the same face of the molecule as the active site cavity, which is open to solvent. C, upper panel: in the presence of Ca²⁺, 15-LOX-2 co-elutes with nanodiscs. Dashed line, 0.5 mm EDTA; solid lines, plus 2 mm CaCl₂. Blue, wild-type; red, D39A/E44A; gray, D39A/E44A/D85A. Lower panel: for reference, individual elution profiles for nanodiscs (ND; black) and 15-LOX-2 (blue); dashed line, 0.5 mm EDTA; solid line, plus 2.0 mm Ca²⁺. D, detail of the Ca²⁺-binding site that anchors the insertion loop. Three main chain carbonyls, Asp⁸⁵, and water (red spheres) coordinate the Ca²⁺ (blue spheres).

FIGURE 3.

The structure was determined in the presence of a competitive inhibitor. A, omit map (calculated without C8E4) electron density (2F_o − F_c, 1σ) positioned above the active site iron is consistent with the detergent C8E4. B, the amino acids that line the active site cavity. Shown is atomic coloring (green, carbon; red, oxygen; blue, nitrogen), with invariant/highly-conserved amino acids in gray. C, non-linear regression analysis of enzyme activity measured at varying inhibitor and substrate concentrations. (♦, 0 mm; ○, 0.1 mm; ▾, 1.0 mm C8E4; AA from 1 to 40 μm). The points represent the observed velocities, and the lines represent the best fit of the data to Equation 2. D, AA (purple and yellow stick renderings; red, oxygen) can be modeled into the C8E4 density in two inversely related orientations. E, detail of the placement of the yellow AA in D. The iron (rust) coordination sphere is filled by His-373, His-378, His-553, the carboxyl terminus, and two water molecules (red). The hydrogen of the pentadiene is poised for abstraction. The asterisk marks C13. The inverse fit of AA C7 sits at this position.

FIGURE 4.

Mechanistic details are consistent with a unique orientation of AA in the active site. A, the contour of the active site with bound C8E4 is complementary to AA in shape, and the catalytic iron and apparent oxygen channel lie on opposite sides of the tunnel. This relationship between O₂ channel and catalytic iron is shown clearly in B. C, schematic of the two fits (Fig. 3D) of AA. The substrate can be modeled it with its tail (top) or head deepest in the cavity (bottom). The depth of the pocket and the orientation of the substrate determine which pentadiene is positioned for attack. In the top orientation, the pentadiene at C13 is positioned for attack; in the lower, C7 is positioned proximal to the red sphere (iron) behind the plane. The blue peanut represents the O₂ channel slightly deeper in the active site but above the plane. A single product is produced by each orientation: with the tail end innermost AA is transformed to 15-S-HPETE, whereas the inverse orientation results in 5-S-HPETE.

FIGURE 5.

The substrate binding cavities of 15-LOX-2 and stable 5-LOX. A, a surface rendering of the inhibitor binding site in 15-LOX-2 reveals a cavity that can accommodate AA. The catalytic iron is behind the inhibitor (orientation similar to that in Figs. 3A and 4A), and an apparent O₂ channel is on the upper face (29). B, a similar rendering of the cavity in 5-LOX with the 15-LOX-2 inhibitor superimposed. Note that the volumes of the cavities beyond the O₂ channel are similar, but that 5-LOX is “corked” above the channel by two amino acids from α2 (Phe¹⁷⁷, Tyr¹⁸¹). C, schematic of inverse orientations of AA (black, tail first). Amino acids that line the cavity in 15-LOX-2 are listed with 5-LOX counterparts in parentheses. Invariant amino acids are in boldface type. Amino acid changes that result in an increase in positive change deep in the cavity of 5-LOX are highlighted in blue.