Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation

Abstract

The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.

Conformational flexibility, lipid binding, and regulatory domains in CelTOS

“…To test this hypothesis, we structurally designed a disulfide-stabilized dimer of CelTOS through incorporation of site-targeted cysteine substitutions. Valle et al used a similar disulfide bridge approach to lock sticholysin I in the homo-dimer state (Valle et al, 2018). The disulfide locked sticholysin I homo-dimer could not form pores and was 193 times less hemolytic than the WT protein (Valle et al, 2018).…”

“…Valle et al used a similar disulfide bridge approach to lock sticholysin I in the homo-dimer state (Valle et al, 2018). The disulfide locked sticholysin I homo-dimer could not form pores and was 193 times less hemolytic than the WT protein (Valle et al, 2018). Similarly, Nguyen et al created double-cysteine LukF (Leukocidin fast fraction) mutants and showed the disulfide-trapped mutants were inactive due to the transition arrest from pre-pore to pore (Nguyen, Higuchi, & Kamio, 2002).…”