Lysine 77 is a key residue in aggregation of equinatoxin II, a pore-forming toxin from sea anemone Actinia equina.
Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 --> Cys influences the normal development of the aggregate which is required for assembling the functional pore.
Anderluh
G
G
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
Barlic
A
A
Potrich
C
C
Macek
P
P
Menestrina
G
G
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003064:Cnidarian Venoms / Q000097:blood / Q000737:chemistry* / Q000633:toxicity*; D003603:Cytotoxins / Q000097:blood / Q000737:chemistry* / Q000633:toxicity; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000633:toxicity; D012615:Sea Anemones; D017550:Spectroscopy, Fourier Transform Infrared
D003064:Cnidarian Venoms; D003603:Cytotoxins; D011994:Recombinant Proteins; C012307:equinatoxin; D008239:Lysine