Membrane fluidity is a key modulator of membrane binding, insertion, and activity of 5-lipoxygenase

Abstract

Mammalian 5-lipoxygenase (5-LO) catalyzes conversion of arachidonic acid to leukotrienes, potent mediators of inflammation and allergy. Upon cell stimulation, 5-LO selectively binds to nuclear membranes and becomes activated, yet the mechanism of recruitment of 5-LO to nuclear membranes and the mode of 5-LO-membrane interactions are poorly understood. Here we show that membrane fluidity is an important determinant of membrane binding strength of 5-LO, penetration into the membrane hydrophobic core, and activity of the enzyme. The membrane binding strength and activity of 5-LO increase with the degree of lipid acyl chain cis-unsaturation and reach a plateau with 1-palmitoyl-2-arachidonolyl-sn-glycero-3-phosphocholine (PAPC). A fraction of tryptophans of 5-LO penetrate into the hydrocarbon region of fluid PAPC membranes, but not into solid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine membranes. Our data lead to a novel concept of membrane binding and activation of 5-LO, suggesting that arachidonic-acid-containing lipids, which are present in nuclear membranes at higher fractions than in other cellular membranes, may facilitate preferential membrane binding and insertion of 5-LO through increased membrane fluidity and may thereby modulate the activity of the enzyme. The data presented in this article and earlier data allow construction of a model for membrane-bound 5-LO, including the angular orientation and membrane insertion of the protein.

FIGURE 1

Activity of 5-LO increases with an increasing degree of lipid cis-unsaturation and decreases in the presence of cholesterol in vesicle membranes. (A) Time dependence of conversion of AA to 5-HPETE, as measured by absorption at 238 nm. The buffer (specified in Materials and Methods) contains 100 μM AA and large unilamellar vesicles composed of 350 μM DPPC (1), POPC + 20 mol % cholesterol (2), POPC (3), PLPC (4), PAPC (5), and PDPC (6). The reaction is initiated by adding 2.4 μg/ml 5-LO, as indicated by the arrow. Measurements are conducted at 22°C. (B) A bar graph showing the mean values and standard deviations of 5-LO activity in the presence of vesicles of different lipid compositions, as indicated. Experimental conditions are as in A. Data shown in B are averaged based on three independent experiments.

FIGURE 2

Dependence of the specific activity of 5-LO on the content of anionic lipids in fluid (POPC/POPG) and solid (DPPC/DPPG) membranes. Total lipid concentration was 350 μM in all cases, AA concentration was 100 μM, and 5-LO was added to a final concentration of 2.4 μg/ml. The buffer, method of 5-LO activity measurement, and other experimental conditions are described in Materials and Methods.

FIGURE 3

Fluorescence emission spectra of 1 mol % Laurdan in vesicles composed of DPPC (1), POPC + 20 mol % cholesterol (2), POPC (3), PLPC (4), PAPC (5), and PDPC (6). The buffer contained 0.1 mM EGTA, 0.3 mM CaCl₂, and 50 mM Tris-HCl (pH 7.5). Total lipid concentration was 100 μM in 0.4-cm path-length quartz cuvettes. The excitation wavelength was 360 nm, and the temperature was 22°C. The inset shows the dependence of GP on the lipid composition, as specified in the main figure. The values of GP were calculated as GP = (F₄₃₅ − F₅₀₀)/(F₄₃₅ + F₅₀₀), where F₄₃₅ and F₅₀₀ are the Laurdan fluorescence emission intensities at respective wavelengths.

FIGURE 4

Fluorescence emission spectra of tryptophans of 5-LO in the absence and presence of large unilamellar PAPC vesicles without (A) and with (B) 2 mol % Py-PE. In both panels, increasing darkness of the lines corresponds to an increase in total lipid concentration from zero to 760 μM (see Fig. 5). Decrease in Trp emission intensity in A is due to dilution upon addition of stock vesicle suspension. In B, Trp fluorescence significantly decreases upon addition of Py-PE-containing vesicles due to energy transfer from Trp of 5-LO to Py-PE. The excitation wavelength was 290 nm, and buffer and temperature were as in Fig. 3.

FIGURE 5

Isotherms of 5-LO binding to vesicles composed of DPPC (•), POPC (▪), PLPC (▴), PAPC (□), and PDPC (▵). The data points are obtained on the basis of the decrease in Trp fluorescence emission intensity due to energy transfer from Trp to Py-PE (2 mol % in vesicle membranes), as measured in RET experiments (e.g., Fig. 4). The theoretical curves are simulated through Eq. 1 using binding parameters summarized in Table 1.

FIGURE 6

Fluorescence emission spectra of tryptophans of 5-LO in the absence and presence of large unilamellar vesicles composed of a fluid lipid, PAPC (A), or a solid lipid, DPPC (B). Only the top portions of spectra are shown to make differences between spectra more discernable. The spectra are numbered as follows: 1, free 5-LO in buffer; 2, 5-LO with large unilamellar vesicles of PAPC (A) or DPPC (B); 3, 6,7-Br₂PC-containing vesicles; 4, 9,10-Br₂PC-containing vesicles; 5, 11,12-Br₂PC-containing vesicles; and 6, POPTC-containing vesicles. Numbering in A also applies to the same line types in B. (C) Summary of the data obtained in three experiments (mean ± SD). Open and solid bars apply to PAPC and DPPC membranes, respectively. When vesicles were present, the total lipid concentration was 750 μM. Brominated lipids were present at 25 mol %, and POPTC at 15 mol %. Protein concentration was 0.24 μM. Excitation was at 290 nm. The buffer and temperature were as in Fig. 3.

FIGURE 7

A model for 5-LO bound to a phospholipid membrane in the fluid phase, constructed on the basis of homology modeling and data on membrane insertion and angular orientation of 5-LO. The protein structure is modeled using SWISS-MODEL on the basis of rabbit 15-LO crystal structure as a template, and is presented in a ribbon format. The N-terminal β-barrel and the catalytic domains are shown in plum and aqua, respectively. The mesh of gray spheres represents the plane of the phosphate groups of phospholipid molecules. Membrane-interacting and catalytically important residues are shown in the ball-and-stick format and are labeled by blue and red labels, respectively. Tryptophans that insert into the membrane hydrocarbon region are shown in yellow, and those located at the membrane-water interface are shown in orange. Phe¹⁹⁷ and Lys¹⁸³, which interact with the membrane by nonpolar and ionic interactions, are shown in green and purple, respectively. Arg⁴¹¹, which presumably forms an ionic contact with the carboxyl group of arachidonate during the initial step of enzyme-substrate interaction, is shown in magenta. Residues involved in coordination of the iron cofactor are colored according to the atom type. Trp¹³, and partially Trp⁵⁹⁹ and Lys¹⁸³, are underneath the polar groups of lipids and can hardly be seen. His³⁶⁷ is hidden behind the helical ribbon. According to our earlier results (Pande et al., 2004), the symmetry axis of the N-terminal β-barrel domain is tilted at ∼45° with respect to the membrane normal, and the current data suggest that at least one Trp, most likely the Trp⁷⁵, inserts into the membrane as deep as 8–9 Å from the membrane center.