Molecular Discrimination between Two Conformations of Sphingomyelin in Plasma Membranes

Abstract

Sphingomyelin and cholesterol are essential lipids that are enriched in plasma membranes of animal cells, where they interact to regulate membrane properties and many intracellular signaling processes. Despite intense study, the interaction between these lipids in membranes is not well understood. Here, structural and biochemical analyses of ostreolysin A (OlyA), a protein that binds to membranes only when they contain both sphingomyelin and cholesterol, reveal that sphingomyelin adopts two distinct conformations in membranes when cholesterol is present. One conformation, bound by OlyA, is induced by stoichiometric, exothermic interactions with cholesterol, properties that are consistent with sphingomyelin/cholesterol complexes. In its second conformation, sphingomyelin is free from cholesterol and does not bind OlyA. A point mutation abolishes OlyA's ability to discriminate between these two conformations. In cells, levels of sphingomyelin/cholesterol complexes are held constant over a wide range of plasma membrane cholesterol concentrations, enabling precise regulation of the chemical activity of cholesterol.

Keywords: ALOD4; cholesterol; cholesterol homeostasis; lipid sensors; ostreolysin A; plasma membrane structure; sphingomyelin; sphingomyelin/cholesterol complexes.

Figure 1.. Lipid binding specificity of SM-sensing proteins

a, SM sensors. Coomassie staining of purified recombinant Lys-His₆, His₆-Eqt, and OlyA-His₆ (2 μg each) b, Lipid specificity. After incubation of SM sensors with liposomes of the indicated compositions for 1 h at room temperature, liposome-bound proteins were measured using a pelleting assay. c, Hemolysis inhibition assay to measure solution binding of SM and cholesterol to OlyA and ALOFL. In each reaction, the indicated lipids were dried on the sides of a tube, after which buffer A containing either OlyA (3 μM) or ALOFL (30 nM) was added. After overnight incubation, PlyB (10 nM) was added to tubes containing OlyA, and then buffer C containing ~3 × 10⁸ RBCs was added to all tubes. Following incubation for 30 min, hemolysis was assayed (n=3). For testing sequential addition, after overnight incubation of proteins with either SM or cholesterol, the contents of assay tubes were transferred to another tube containing dried cholesterol or SM, respectively, and the remainder of the assay was carried out as above. d-e, Dependence of OlyA binding on membrane lipid mobility. d, Supported bilayers of the indicated lipid compositions containing TR-DHPE (fluorescent lipid) were generated in glass-bottom 96-well plates and lateral fluidity of lipid molecules was measured by FRAP before and after fixation with osmium tetroxide. Gray bar denotes the 30s photobleaching step. Shown are averages of fluorescence values from three different regions in a well. e, Unfixed and osmium tetroxide-fixed supported bilayers of the indicated lipid composition were incubated with 5 μg of fOlyA for 30 min, after which membrane-bound fOlyA and TR-DHPE was measured (n=4). f-g, Dependence of OlyA binding on SM:cholesterol ratio. f, Binding to liposomes containing SM and varying molar concentrations of the indicated sterol was measured as in b, except that the incubation time was 4 h (n=3). g, Binding to lipid films. Ethanolic solutions of varying molar ratios of SM and the indicated sterol were prepared and 80 nmol of each mixture was deposited on nitrocellulose membranes and allowed to dry for 5-10 min. Each membrane strip was then subjected to dot blot analysis to measure bound OlyA (n=3). Chol., cholesterol; Epi., epicholesterol.

Figure 2.. Structural analysis of SM binding by wild-type OlyA

a, Overall structure of OlyA (teal) bound to a portion of SM (yellow sticks, see c). Possible orientations for the rest of OlyA-bound SM are shown as yellow ovals. b, Close-up view of a surface representation of the shallow channel formed by K99 and W28 (boxed region in a) which houses the observed electron density (gray mesh, see c). Superimposed on the density is the modeled portion of SM (yellow sticks, see c). c, Shown in gray mesh is the ∣mFo – DFc∣ electron density calculated after omitting the ligand from the model and contoured at 2.5σ. A portion of 18:1 SM (highlighted in red) was superimposed on this electron density. d, Close-up view of the region of OlyA with bound SM. Side-chains of amino acids that lie within 5 Å of the modeled portion of SM (yellow) are shown as sticks (teal) against a semi-transparent main chain backbone (light teal). e, Binding properties of OlyA mutants. Indicated His₆-tagged mutant versions of OlyA were overexpressed, purified, and their lipid specificities were measured using liposome dot blot assays. The mean value (n = 3) for binding of OlyA(WT) to 1:1 SM:cholesterol liposomes was set to 1. All other mean binding values (n = 3) were normalized relative to this set-point and converted to a green-to-red color scale. Standard errors for all measurements were less than 10%. Chol., cholesterol; Epi., epicholesterol; N, NH₂-terminus; C, COOH-terminus.

Figure 3.. Structural analysis of SM binding by OlyA(E69A)

a, Effects of mutation of E69 on OlyA’s lipid specificity. Indicated His₆-tagged mutant versions of OlyA were overexpressed, purified, and their lipid specificities were measured using liposome dot blot assays. The mean value (n = 3) for binding of OlyA(WT) to 1:1 SM:cholesterol liposomes was set to 1. All other mean binding values (n = 3) were normalized relative to this set-point and converted to a green-to-red color scale. Standard errors for all measurements were less than 10%. b, Overall structure of OlyA(E69A) (purple) bound to bis-tris (yellow sticks, see d). c, Close-up view of a surface representation of the shallow channel formed by K99 and W28 (boxed region in b) which houses the observed electron density (gray mesh, see d). Superimposed on the density is the modeled bis-tris structure (yellow sticks, see d). d, Shown in gray mesh is the ∣mFo – DFc∣ electron density calculated after omitting the ligand from the model and contoured at 2.5σ. The structure of bis-tris (highlighted in red) was superimposed on this electron density. e, Overlay of the regions containing bound ligands in OlyA(WT) (teal) and OlyA(E69A) (purple). Side-chains of amino acids that have different orientations in the two structures are shown as sticks against a semi-transparent main chain backbone. Chol., cholesterol; Epi., epicholesterol; N, NH₂-terminus; C, COOH-terminus.

Figure 4.. Comparison of affinities and lipid specificities of OlyA(WT) and OlyA(E69A)

a, Concentration dependence. Binding of OlyA(WT) and OlyA(E69A) to liposomes of the indicated compositions was measured by dot blot assays (1 μg/ml of OlyA = 57.6 nM). The mean value (n = 3) for binding of each protein to liposomes composed of 1:1 SM:Chol. at the highest protein concentration was set to 100% and all other mean binding values (n = 3) were normalized relative to these set-points. b, Association rates. After incubation of fOlyA(WT) and fOlyA(E69A) with liposomes of the indicated compositions for various times, liposome-bound fOlyA was measured. 100% of control values for fraction of bound proteins were 0.33 for fOlyA(WT) and 0.604 for fOlyA(E69A) (n=3). c-d, Phospholipid and sterol specificity. Dot blot assays were used to measure the binding of OlyA(WT) and OlyA(E69A) to liposomes composed of equimolar mixtures of cholesterol and phospholipids with 18:1 acyl chains and the indicated headgroup (c, left), cholesterol and SM with the indicated amide-linked acyl chain length (c, right), or 18:1 SM and the indicated sterol (d). The mean value (n = 3) for binding of each protein to liposomes composed of cholesterol and 18:1 SM was set to 100%. All other mean binding values (n = 3) were normalized relative to this set-point. e, Temperature dependence. After incubation of OlyA(WT) and OlyA(E69A) with liposomes of the indicated compositions for 4 h at various temperatures, liposome-bound OlyA was measured using a pelleting assay (centrifugation steps were also carried out at various temperatures). 100% of control values for fraction of bound proteins were 0.604 for OlyA(WT) and 0.704 for OlyA(E69A) (n=3). Epi., epicholesterol; Dchol., dihydrocholesterol; Chol., cholesterol.

Figure 5.. Docking simulations for binding of SM to OlyA(WT) and OlyA(E69A) and chemical modification assays

a, Chemical structure of 18:1 SM with the fragment used in simulations highlighted (red). b – e, Top-scoring models are shown for binding of SM (yellow spheres) to OlyA(WT) (b) and OlyA(E69A) (d). Plausible schematic models for binding of OlyA(WT) (c) and OlyA(E69A) (e) to SM in membranes are shown with proteins depicted as cartoons with transparent surfaces and the top scoring docking poses of bound SM (yellow sticks) oriented on the membrane surface with acyl chains extrapolated into membrane bilayer (yellow ovals). f, g, Chemical modification of OlyA(WT) and OlyA(E69A). OlyA(WT)-His₆ (f) or OlyA(E69A)-His₆ (g) were incubated with indicated liposomes for 3h at room temperature, after which liposome-bound OlyA proteins were subjected to modification with mPEG-MAL-5000 followed by immunoblot analysis. As controls, OlyA(WT)-His₆ or OlyA(E69A)-His₆ in solution were also subjected to mPEG-MAL-5000 modification. M, modified form of OlyA; U, unmodified form of OlyA. h, Schematic description of mPEG-MAL-5000 modification results from f and g.

Figure 6.. Organization of SM and cholesterol in PMs of CHO-K1 cells

a, Dependence of OlyA binding on membrane lipid mobility. Lateral fluidity of lipid molecules in CHO-K1 cell membranes was measured by FRAP before and after fixation with osmium tetroxide (left). Gray bar denotes the 30s photobleaching step. Shown are averages of fluorescence values from three different regions in a well. Unfixed and osmium tetroxide-fixed wells were incubated with the indicated fOlyA (3 μM) for 30 min, after which membrane-bound fOlyA and TR-DHPE was measured (right) (n=4). b-d, On day 0, CHO-K1 cells were set up in medium B at a density of 2.5 × 10⁵ cells/60-mm dish (b, 6 dishes/replicate/condition; c-d, 12 dishes/replicate/condition). b, SMase treatment. On day 2, cells were switched to fresh medium B containing the indicated concentrations of SMase. After incubation for 30 min at 37°C, cells were washed and 2 dishes from each cond ition were incubated with 3 μM of the indicated sensor protein. After incubation for 1 h at 4°C, cells were harvested and PM-bound proteins were quantified. (n=3) c, Cholesterol modulation by serum and compactin. On day 1, cells were switched to either fresh medium B, medium C, or medium D containing the indicated serum without or with compactin. On day 2, cells were washed and binding of the indicated sensor proteins was carried out as in b for 6 dishes/replicate/condition. Cells from the remaining 6 dishes were pooled and used for PM purification and cholesterol quantification. (n=3) d, Cholesterol modulation by cyclodextrin. On day 2, cells were switched to fresh medium B (one group) or fresh medium C containing 0.01% - 2% (w/v) HPCD. After incubation for 1 h at 37°C, cells were washed and b inding of the indicated sensor proteins and quantification of PM cholesterol was carried out as in c (n=3). 100% of control values for bound ALOD4, OlyA(WT), and OlyA(E69A) were 5.7, 3.7, and 5.4 μg/mg protein, respectively. e, On day 0, the indicated cell lines were set up at a density of 2.5 × 10⁵ cells/60-mm dish (media described in Methods; 6 dishes/replicate/condition). On day 1, cells were switched to media containing the indicated serum without or with compactin. On day 2, some cells were further treated with SMase (100 mU/mL) or HPCD (1% w/v) as described above, after which all cells were washed and binding of 3 μM of the indicated sensor protein was carried out as described above. 100% of control values for bound ALOD4, OlyA(WT), and OlyA(E69A) for each cell line was as follows: SV-589 (4.9, 3.5, and 6.8 μg/mg protein); Neuro-2A (1.1, 1.8, and 2.6 μg/mg protein); ST88-14 (6.3, 14.3, and 18.2 μg/mg protein); MDCK (4.3, 3.8, and 5.2 μg/mg protein); Caco-2 (4.5, 8.3, and 10.2 μg/mg protein) (n=3).