Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15

Abstract

Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties.

Keywords: cooperative effects; crystal structure; lipoxygenases; molecular dynamics; protein–protein interactions.

Figure 1

(A) Overlay of crystal structures of rabbit ALOX15 (PDB entry 2P0M, conformer A, grey) and human ALOX15B (PDB entry 4NRE, brown). (B) Overlay of crystal structures of rabbit ALOX15 (PDB entry 2P0M, conformer A, grey) and the AA 12-lipoxygenating porcine ALOX15 catalytic domain (PDB entry 3RDE, brown). (C) Structural heterogeneity of rabbit ALOX15 and human ALOX15 B in salt-free aqueous solutions. (C-I) Native polyacrylamide gel electrophoresis (PAGE). For this analysis, the enzymes were prepared and maintained in 20 mM Tris-HCl buffer, pH 8.0 containing 130 mM NaCl. (C-II) Native PAGE. For this analysis, the enzyme was prepared and maintained in 20 mM Tris-HCl buffer, pH 8.0 that does not contain NaCl (desalted enzyme preparation eluted as a single peak by and size-exclusion chromatography). (C-III) Denaturing PAGE. For this analysis the enzyme was prepared and maintained in 20 mM Tris-HCl buffer, pH 8.0, but the running buffer contained 0.5% SDS.

Figure 2

Structural characterization of wild-type and mutant rabbit ALOX15. (A) Native PAGE electrophoresis of rabbit ALOX15 and its catalytic domain. (B) Effect of pH on structural heterogeneity of ALOX15. At pH 8.0 the two conformers (red labelled enzyme pools I and II) are better resolved when compared with pH 6.8. (C) Crystal structure of conformer A (no ligand at the active site). In this structure the side chain of His585 (H585) is localized on the protein surface and is accessible to the solvent. (D) Crystal structure of conformer B (ligand bound at the active site). Here the side chain of His585 (H585) is buried inside the protein between the side chains of Glu185 (E585) and Lys189 (K189). It is shielded from the solvent. (E) Elution order of the purified ALOX15 variants from the Resource Q (6 mL) column in anion exchange chromatography using a linear NaCl gradient. Absorbance of the column effluent at 280 nm (green, black, blue) and conductivity (red curve) were simultaneously recorded. (F) Size exclusion chromatography (left panel) of the purified recombinant proteins was carried in 20 mM Tris-HCl buffer and we estimated their hydrodynamic radii using different calibration proteins (right panel). The elution volume of the wild-type ALOX15 and the mutants is labelled with asterisk. (G) Native PAGE of rabbit ALOX15 mutants was performed at two different pH as described above for the wild-type ALOX15. (H) SDS-PAGE of the enzyme preparations (the numbers correspond to the protein samples that are present on panels B and G). (I) CD spectra of wild-type and mutant rabbit ALOX15. (J) Thermal stability of wild-type rabbit ALOX15 and its His585Glu (H585E) mutant. The relative intensity of the CD signal of ALOX solution was measured at 220 nm at different temperatures. (K) Fluorescence spectra of wild-type and mutant rabbit ALOX15. (L) The shift of the maximum of the fluorescence spectrum in the presence of different concentrations of GdnHCl was monitored by fluorescence steady state spectroscopy for wild-type rabbit ALOX15 and its His585Glu (H585E) mutant. The GdnHCl denaturation curves follow a 3-state transition model (ground state M ↔ intermediate sate I ↔ unfolded state U).

Figure 3

Contribution of Trp181 (W181) and His585 (H585) to the inter-monomer interface of rabbit ALOX15 and impact of mutations on the reaction kinetics of the enzyme. (A) Crystal structure of the rabbit ALOX15 heterodimer (PDB entry 2P0M) consisting of a ligand-free conformer A (brown) and a ligand-bound conformer B (grey). Inset: Amino acid residues contributing to the inter-monomer interface. (B) The two structures of the rabbit ALOX15. α2 Helix of conformer A (α2A) is strongly dislocated when compared with conformer B (α2B). (C) Reaction kinetics of wild-type rabbit ALOX15 and of two enzyme mutants. Linoleic (left panel) or arachidonic acid (right panel) oxygenation was assayed spectrophotometrically (increase in absorbance at 235 nm) at different substrate concentrations. For each measurement 56 nM LOX (final enzyme concentration) normalized to the iron content was used.

Figure 4

Structural consequences of Trp181Glu exchange on dimer formation and substrate alignment. (A) Inter-monomer interface of wild-type rabbit ALOX15. Secondary structural elements of conformer A are shown in mustard and those of conformer B are given in green. (B). Inter-monomer interface of the Trp181Glu mutant of rabbit ALOX15. Secondary structural elements of conformer A are shown in light blue and those of conformer B in purple. (C) Most representative binding mode of AA in WT-ALOX15 (dark green) and in Trp181Glu-ALOX15 (purple) dimers. WT-ALOX15 (green with a percentage of transparency) and Trp181Glu-ALOX15 (light blue with a percentage of transparency) backbones have been superimposed. The side-chains of some selected residues for WT-ALOX15 and Trp181Glu-ALOX15 dimers have been displayed in green and light blue, respectively. (D) Most representative binding mode of LA in WT-ALOX15 (dark green) and in Trp181Glu-ALOX15 (purple) dimers. WT-ALOX15 (green with a percentage of transparency) and Trp181Glu-ALOX15 (light blue with a percentage of transparency) backbones have been superimposed. The side-chains of some selected residues for WT-ALOX15 and Trp181Glu-ALOX15 dimers have been displayed in green and light blue, respectively. (E) C₁₃-OH distance in the Trp181Glu-ALOX15-AA (in blue) and in the WT-ALOX15-AA (in green) complexes versus time. (F) C₁₁-OH distance in the Trp181Glu-ALOX15-LA (in blue) and in the WT-ALOX15-LA (in green) complexes versus time.