Sphingomyelin SM is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II EqtII.
The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup.
Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp and Tyr exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.
Sphingomyelin SM 8 is an important eukaryotic membrane lipid, located for the most part in the outer leaflet of the plasma membrane in the form of specialized cholesterol-rich microdomains, so-called lipid rafts 1 , 2. Many pathogens and toxic proteins employ lipid rafts to invade cells 3 , 4 , but currently little is known about the molecular details of the recognition mechanism of the lipid components present in the rafts. In the particular case of SM, the specific recognition occurs even though SM exposes the same phosphorylcholine headgroup as the other abundant lipid, phosphatidylcholine.
SM-binding proteins are currently exploited as specific markers for cellular SM 5 and are used to identify other proteins involved in sphingolipid metabolism 6. Actinoporins are extremely potent cytolysins produced exclusively by sea anemones 7 , 8. They may be used to capture prey, in intraspecific aggression, or in preventing adhesion of other organisms 7 , 9.
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Actinoporins constitute a family of conserved proteins that cause hemolysis of red blood cells by colloid-osmotic lysis and exhibit cytolytic activity against various cell lines 7 , 10 — The N-terminal helix is used for transmembrane pore formation 16 , Thus the archetypical actinoporin structural scaffold is widespread and is used for the specific binding to various molecules of the plasma membrane The membrane-lytic activity of actinoporins is highly SM-dependent reviewed in Ref.
However, some published data indicate that the addition of cholesterol to phosphatidylcholine PC liposomes also increases actinoporin permeabilizing activity 26 — It thus remains to be determined whether the SM effect originates from a general influence of the physico-chemical properties of the membrane or is a result of specific binding. Lipid specificity has been poorly addressed in the available structural studies of actinoporins 13 — 15 , 29 , In this work we have tested the hypothesis that EqtII can specifically bind sphingomyelin both in solution and in lipid membranes.
We additionally show that the membrane-binding step is driven mainly by hydrophobic rather than electrostatic interactions and that the residues Trp and Tyr enable SM dependence. Direct binding to SM. A and B , dot-blot binding assay using some of the most common glycerolipids and sphingolipids. The spots contain pmol of lipid A. A commercial membrane was used in B , with lipids as specified by the producer.
Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer.
The first spot contains pmol of lipid, followed by five successive 2-fold serial dilutions. C , binding of lipid analogues by protein as measured by surface plasmon resonance. The curves were normalized to show the binding to RU of immobilized protein. Bovine brain SM was used in all experiments unless stated otherwise. All other chemicals were from Sigma unless stated otherwise. Recombinant EqtII, mutants and histidine-tagged variants were prepared in Escherichia coli as described previously 19 , The tryptophan fluorescence and far UV circular dichroism spectra showed that mutations did not cause large structural changes.
All reported lipid ratios are mol:mol ratios. Liposome Preparations —Large unilamellar vesicles LUV of nm diameter were prepared by extrusion of multilamellar vesicles as described previously When using calcein, excess dye was removed by gel filtration through a small G column. Permeabilization assays of calcein-loaded liposomes were conducted with a Jasco FP spectrofluorometer in a 1.
Proteins Membrane Binding and Pore Formation
The excitation wavelength was set to nm, emission was followed at nm, and both slits were set to 5 nm. Permeabilization was expressed as the percentage of maximal permeabilization obtained at the end of the assay by addition of detergent Triton X to a final concentration of 2 m m. Hemolysis and Preparation of Erythrocyte Ghosts —Hemolysis of human or bovine red blood cells was measured at room temperature by using a microplate reader MRX, Dynex. Ghosts were washed with the same buffer until all hemoglobin was removed, i. SM was removed from erythrocytes by incubation with Bacillus cereus sphingomyelinase.
Erythrocytes were washed extensively before use in hemolytic assay or ghost preparations. The removal of cholesterol and SM from erythrocyte membranes was confirmed by enzymatic tests for lipid concentration determination: phospholipids B for choline-containing lipids and free cholesterol C for cholesterol both from Wako. Additionally, total lipid extracts were prepared from erythrocytes by the method of Bligh and Dyer 33 and analyzed by thin layer chromatography TLC to confirm both the removal of SM and cholesterol and that the amount of other lipids in the membrane was not altered.
An L1 chip was equilibrated with vesicle buffer, and a liposome-coated chip surface was prepared as described previously Proteins 0. Erythrocyte ghosts were immobilized by using the same protocol to a final value of response units RU. The concentration of EqtII was 50 n m for the binding assay employing erythrocyte ghosts.
Blank injections were subtracted from sensorgrams to correct for the buffer contribution. The amount of stably inserted protein after 4 min of dissociation was determined from the sensorgrams and used to report in Figs. Kinetic analysis of EqtII and mutant binding was performed as described above.
Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin
Protein concentrations were adjusted so that the response did not exceed RU. This enabled us to monitor only the initial binding to the liposomes and not the subsequent steps in the pore-forming mechanism The data were globally fitted to a two-step binding model described in detail in Hong et al. The first and last 10 s of the injection and the first 10 s of the dissociation were not included in the fit because of bulk refractive index changes and mixing effects Binding of lipid analogues to proteins was performed on a Biacore Biacore AB.
Association was followed for 1 min and dissociation for 3 min. Lipid analogues were dissolved as 50 m m stock solutions in SPR buffer. Lipid Monolayer Assay —The increase in the surface pressure of lipid monolayers of different compositions was measured with a MicroTrough-S system from Kibron Helsinki, Finland at room temperature.
The increment in surface pressure versus time was monitored until a stable signal was obtained. Sphingo Array, a commercial membrane spotted with lipids as specified by the producer Product No. SC, Echelon , was also used.
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Histidine-tagged protein at final 2. The dot-blot was developed by using 4-chloronaphthol as a substrate. The fluorescence of SYPRO Orange increases upon unfolding of the protein, and hence reports on changes in protein fold and stability are made possible. Structural figures were prepared with PyMol We first performed a lipid dot-blot assay with a range of sphingolipids and some other lipids that were used in this study Fig.
EqtII recognized only two different sphingomyelins, i. These two differ in the properties of the fatty acid acyl chain but possess the same headgroup. EqtII did not bind to any other lipid tested, most notably cholesterol, phosphatidylcholine, and any of the other sphingolipids used, i.
It also did not bind other sphingolipids and glycerol-based phospholipids not presented in Fig. In summary, Fig. Conformational stability and inhibition of EqtII hemolysis in the presence of lipid analogues. A , thermograms of EqtII alone filled squares and in the presence of phosphocholine open circles and Ac-SM open squares. The arrow indicates the pretransition unfolding peak observed in the presence of Ac-SM.
AU , arbitrary units. B , hemolysis of bovine red blood cells was measured in the presence of lipid analogues at room temperature in m m NaCl, 20 m m Tris-HCl, pH 7. Hemolysis was monitored at nm for 20 min at room temperature. As evident from the superposed thermal scans, phosphocholine does not influence the melting behavior of EqtII peak at In addition to the main transition, which shifts to a slightly lower temperature peak at The stability and hence the compactness of the EqtII structure is clearly reduced in the presence of Ac-SM compared with controls.
Notably, the bimodal melting profile is indicative of a conformational change of EqtII upon Ac-SM binding, which phosphocholine alone is not able to induce. Some previous studies have used many different lipids to assess the interaction with EqtII or sticholysin by measuring the inhibition of permeabilizing activity.
Among lipids used in our binding assays, these studies also employed some other sphingolipids such as cerebrosides 25 , 40 , They showed that only SM was able to considerably inhibit permeabilizing activity. Thus our direct binding studies are in excellent agreement with previous inhibitory tests.
The presence of SM in liposomes enables binding and permeabilization. The concentration of EqtII was n m. The permeabilization induced by EqtII was expressed as the percentage of maximal permeabilization obtained at the end of the assay by the addition of detergent Triton X to a final concentration of 2 m m.
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The amount of stably bound EqtII after 4 min of dissociation is shown for the comparison by solid columns. The experimental conditions were as in A. D , the amount of stably bound EqtII after 4 min of dissociation. These differences in binding might arise from SM-induced changes in the bilayer physical properties, i.
Hence we replaced SM with DPPC, a PC with disaturated acyl chains and physical properties similar to those of SM and the ability to induce lipid domain formation in binary and ternary mixtures 42 , We have also checked binding to DOPC:SM:cholesterol , a ternary lipid mixture that should allow the formation of lipid domains 42 , The binding to these membranes was stable and irreversible, but replacing SM with DPPC again reduced binding dramatically.
We further checked how the presence of different sphingolipids affects binding. As expected, the binding to either porcine brain or chicken egg SM was significant and irreversible, whereas it was negligible for all other sphingolipids tested Fig. Finally, we examined how the composition of red blood cell membranes affects the binding and hemolytic activity of EqtII. The removal of accessible SM from human erythrocyte membranes by sphingomyelinase Fig. We tested whether this lack of hemolytic activity was due to a reduced binding to erythrocyte membranes.
Similarly, we could only detect poor and reversible binding to SM-depleted erythrocyte ghost membranes in SPR experiments. In contrast, untreated and cholesterol-depleted ghost membranes bound EqtII in an irreversible manner Fig. A , thin layer chromatography of lipid extracts from red blood cells used in this experiment.
Lane 1 , cholesterol CHO -depleted erythrocytes; lane 2 , normal erythrocytes; lane 3 , SM-depleted erythrocytes. The position of cholesterol or SM standards is denoted at the side of the image.