A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex

Abstract

A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8 R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca (2+)-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca (2+)-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions.

Fig 1

Size exclusion chromatography of 8R-LOX and AOS. Protein elution was simultaneously monitored at 280 (black) and 406nm (gray). Absorbance at 406nm is due to the AOS heme. (a) HD (LOX:AOS) elutes as a complex at 750s. (b) AOS in excess (2AOS:1LOX) yields two clearly resolved peaks: HD (750s) and AOS (820s). HD has 1 heme for 1066 amino acids, and the absorbance at 280nm is greater than that at 406nm. The reverse is true for AOS, which has 1 heme for 373 amino acids. (c) A 2:1 mixture of AOS: LOX(D411A). The mutation in the LOX C2-like domain impairs HD formation: Note in panel (c) the absence of distinct HD and AOS peaks, an indication that a heterodimer of LOX(D411A)+AOS is not stable in the size exclusion chromatography column.

Fig. 2

Small Angle X-ray Scattering of HD and FP. (a) Radii of gyration and the forward scattered intensity divided by protein concentration recorded for FP and HD as a function of concentration. (b) Composite X-ray scattering curves and corresponding electron pair distance distribution function (inset). Those curves for FP are shown in red, and HD in blue. Also shown in green is the scattering curve predicted for a mixture of LOX and AOS in 1:2 molar ratio without forming heterodimer, based on corresponding crystallographic structures. The same FP curve (scaled at 0.1x) is reproduced with experimental error bars in the lower part of the figure along with the predicted X-ray scattering curve (black, scaled at 0.1x)) for one of the two low-resolution crystal structures of FP.

Figure 3

SAXS-supported crystal structure of FP. (a) A cartoon and surface rendering of FP. The AOS domain is colored N→ C (blue → red). The C2-like and catalytic sub-domains of the LOX portion are in brown and beige, respectively. The gray spheres mark the positions of the Ca²⁺ atoms in the 3.2 Å resolution structure of the LOX domain alone (2FNQ). Neither the Ca²⁺ ions or the putative membrane insertion loops that they stabilize (one of which includes D411, position marked by red oval) are observed with confidence in the FP structure. (b) Detail of the structural elements that make up the AOS-LOX interface. In this rendering, which is identical in orientation as (a), the LOX domain is also colored N→ C (blue → red). Amino terminal AOS helix α2 (blue) is flanked by C-terminal helices of both the AOS and LOX domains (red). Amino acid ligands for the catalytic irons of both domains are directly (AOS) or indirectly (LOX) connected to these helical segments.

Figure 4

Membrane binding monitored by FRET. (a) Pyrene-heme FRET. Emission spectra for pyrene-labeled liposomes and fusion protein (FP, left), heterodimer (HD, middle), and AOS (right). Protein was added to solutions of LUV in which pyrene-labeled phospholipid was incorporated. Emission spectra were recorded for triplicate samples in three states: protein only (solid black), plus CaCl₂ to a final concentration of 2mM (dashed line), and plus EDTA to a final concentration of 4 mM (solid gray). For FP, the addition of CaCl₂ quenches the observed fluorescence, and the subsequent addition of EDTA restores the signal (the spectra are not corrected for dilution.) In contrast, no difference is observed for either the HD or AOS samples in the three states. Thus heme, the prosthetic group of AOS, is only localized to the membrane by Ca²⁺ when covalently bound to LOX. (b) AOS titration of Ca²⁺-dependent membrane binding by 8R-LOX. Emission spectra for 8R-LOX alone (0.5 μM, solid black) and in the presence of increasing AOS (from 0.16 to 1.3 μM). Ca²⁺-dependent binding of 8R-LOX to LUV can be measured by FRET when dansyl-labeled phospholipids are incorporated into the vesicles (dLUV (11)). Titration of the sample with AOS results in a quench of the dansyl signal.

Fig 5

Protein pelleting with sucrose-loaded LUV’s. (a) An amount equivalent to the total protein in the incubation mixture applied to an SDS-PAGE gel. (b) Protein pelleted with sucrose-loaded LUV as a function of Ca²⁺ concentration. Protein and sucrose-loaded LUV, at concentrations of CaCl₂ from 0-1mM, were centrifuged at 100,000 × g for 1 hr. Membrane fractions were applied to a 10 % SDS-PAGE and the protein visualized by Coomassie staining. (c) A plot of the intensity of the protein bands vs. CaCl₂ concentration. Free AOS (dotted line) does not translocate to the membrane in the presence of its partner protein LOX.

Fig. 6

Accessibility to the TEV-protease Cleavage Site. (a) Cartoon of the model for membrane binding by the FP. The TEV protease site is positioned in the linker between the AOS and LOX domains (indicated by *). Ca²⁺-dependent membrane binding releases AOS from the non-covalent interactions and results in improved access the TEV-cleavage site by protease. (b) TEV-FP was incubated with TEV protease in buffer plus LUV prepared from 3:1 PC:PS (LUV) or 3:1 AA-PC:PS (ALUV) in the presence or absence of 2 mM CaCl₂. A parallel incubation was performed with Dodecylmaltoside (300 μM) in place of LUV. The reaction was stopped after 4 hours and the products separated by SDS-PAGE and visualized with Coomassie stain. (c) A plot of the integrated LOX and AOS band intensities for the gel in (b). Baseline levels of LOX and AOS are observed in the presence of LUV but absence of Ca²⁺ (lanes 1 and 2) or in the presence of Ca²⁺ and detergent (lane 5). The addition of Ca²⁺, which promotes membrane binding of TEV-FP, results in an increase in the LOX and AOS produced. When the liposomes contain AA-PL, the stimulation is enhanced. Thus the phospholipid content impacts TEV-protease susceptibility only in the presence of Ca²⁺, conditions which promote membrane binding by of FP.