Interaction of the Pore-Forming Protein Equinatoxin II with Model Lipid Membranes:  A Calorimetric and Spectroscopic Study

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the fulllength toxin is consistent with two endothermic transitions. Whereas peak A (T m ‫؍‬ 55°C) most likely corresponds to the receptor/translocation domain, peak B (T m ‫؍‬ 59°C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18°C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T m ‫؍‬ 65°C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B (Ϸ40%) or its pore-forming fragment (Ϸ80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T m ‫؍‬ 54°C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.

Section: Structural and Functional Characteristics Of Colicin B Andsupporting

confidence: 91%

Calorimetric Investigations of the Structural Stability and Interactions of Colicin B Domains in Aqueous Solution and in the Presence of Phospholipid Bilayers

Ortega¹,

Lambotte²,

Bechinger³

2001

“…5C) with an abundance of positive charge around the IBS. It has already been proposed that this positive charge may help in the initial contact of EqtII with the membrane (65). However, this structural feature is not conserved among actinoporins, and it seems that electrostatic interactions are not important for the specific recognition of SM-containing membranes and other interactions are predominant.…”

Section: Discussionmentioning

confidence: 97%

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Bakrač

Gutiérrez-Aguirre

Podlesek

et al. 2008

Self Cite

165

169

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 112 and Tyr 113 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). The two most studied representatives are EqtII, isolated from the sea anemone Actinia equina, and sticholysin II (StII) from Stichodactyla helianthus. 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 -12). Even the most distant members of the family share more than 60% sequence identity and the available threedimensional structures of EqtII and StII are nearly superimposable (13-15). The structure is composed of a tightly folded ␤-sandwich flanked b...

“…Binding of equinatoxin II to SM is, however, not specific. Equinatoxin II also interacts with phosphatidylcholine and phosphatidylglycerol under appropriate conditions (28,30). Sticholysin I and II from Stichodactyla helianthus also prefer SM-containing membranes (31) and Vibrio cholerae cytolysin requires both SM and cholesterol (32).…”

Section: Discussionmentioning

confidence: 99%

“…If lysenin interacts with hydrophobic tails, it disturbs cooperative phase transition of the lipid and the enthalpy of phase transition is decreased (27,28). In this experiment, we employed synthetic phospholipids with defined phase transition temperature: C16:0 SM (phase transition at 37.3°C) and diC16:0 PC (phase transition at 41.5°C).…”

Section: Lysenin Assembles To Sds-resistant Oligomers In the Presencementioning

confidence: 99%

Oligomerization and Pore Formation of a Sphingomyelin-specific Toxin, Lysenin

Yamaji‐Hasegawa

Makino

Baba

et al. 2003

121

146

Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of ϳ3 nm. Antibody scanning analysis suggested that the Cterminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.