Lysine 77 is a key residue in aggregation of equinatoxin II, a pore-forming toxin from sea anemone Actinia eguina

Among eighteen point mutants of equinatoxin II produced in E. coli, contain ing a single cystein substitution at variable position, EqtIIK77C was chose n for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical mod ification of the thiol group, provided that a positive charge was reintrodu ced. This indicates that a positive charge at this position is necessary fo r toxin activity. The mutant formed larger pores as compared to the wild ty pe, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtII K77C and its binding to lipid membranes, either vesicles or red blood cells , was almost normal. However the kinetics of calcein release from lipid ves icles was substantially slower than that of the wild-type. Taken together w ith the different size of the pore formed, this is an indication that mutat ion of Lys77 --> Cys influences the normal development of the aggregate whi ch is required for assembling the functional pore.