THE NOVEL ACIDOPHILIC STRUCTURE OF THE KILLER TOXIN FROM HALOTOLERANTYEAST DEMONSTRATES REMARKABLE FOLDING SIMILARITY WITH A FUNGAL KILLERTOXIN

Background: Several strains of yeasts and fungi produce proteinous sub stances, termed killer toxins, which kill sensitive strains. The SMK t oxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, u niquely exhibits its maximum killer activity under conditions of acidi c pH and high salt concentration. The toxin is composed of two distinc t subunits, alpha and beta, which tightly interact with each other und er acidic conditions. However, they are easily dissociated under neutr al conditions and lose the killer activity. The three-dimensional stru cture of the SMK toxin will provide a better understanding of the mech anism of toxicity of this protein and the cause of its unique pH-depen dent stability. Results: Two crystal structures of the SMK toxin have been determined at 1.8 Angstrom resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-s andwich family. The folding topology of the SMK toxin is essentially t he same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split beta alpha beta motifs, which are rare in the other proteins. Many acidic residues are clustered at the botto m of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electros tatically bound near the basic residues which are also locally distrib uted at the bottom of the toxin molecule. Conclusions: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stabil ity of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineerin g, for instance, towards stabilization of the toxin molecule in a broa der pH range. The present crystallographic study revealed that the str ucture of the SMK toxin itself is hardly affected by the ionic strengt h, implying that a high salt concentration affects the sensitivity of the cell against the toxin.