2NH and 3OH are crucial structural requirements in sphingomyelin for sticholysin II binding and pore formation in bilayer membranes
Sticholysin II (StnII) is a pore-forming toxin from the sea anemone Stichodactyla heliantus which belongs to the large actinoporin family. The toxin binds to sphingomyelin (SM) containing membranes, and shows high binding speci?city for this lipid. In this study, we have examined the role of the hydrogen bondinggroups of the SM long-chain base (i.e., the 2NH and the 3OH) for StnII recognition. We prepared methylated SM-analogs which had reduced hydrogen bonding capability from 2NH and 3OH. Both surface plasmon resonance experiments, and isothermal titration calorimetry measurements indicated that StnII failed to bind to bilayers containing methylated SM-analogs, whereas clear binding was seen to SM-containing bilayers. StnII also failed to induce calcein release (i.e., pore formation) from vesicles made to contain methylatedSM-analogs, but readily induced calcein release from SM-containing vesicles. Molecular modeling of SM docked to the phosphocholine binding site of StnII indicated that the 2NH and 3OH groups were likely to form a hydrogen bond with Tyr135. In addition, it appeared that Tyr111 and Tyr136 could donate hydrogen bonds to phosphate oxygen, thus stabilizing SM binding to the toxin. We conclude that the interfacial hydrogen bonding properties of SM, in addition to the phosphocholine head group, are crucial for high-af?nity SM/StnII-interaction.
a) Biochemistry, Department of Biosciences, Åbo Akademi University, Turku, Finland
b) Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain
c) Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland
d) School of Science & Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda City, Hyogo 669-1337, Japan