Crystal Structure of Crotoxin Reveals Key Residues Involved in the Stability and Toxicity of This Potent Heterodimeric β-Neurotoxin

Abstract

The crystal structure of crotoxin, a potent presynaptic neurotoxin from Crotalus durissusterrificus, was solved at 1.35 Å resolution. It shows the architecture of the three disulfide-linked polypeptide chains (α, β, and γ) of the acidic subunit CA noncovalently complexed with the basic phospholipase A2 (PLA2) subunit CB. The unique structural scaffold of the association of the CA and CB subunits indicates that posttranslational cleavage of the pro-CA precursor is a prerequisite for the assembly of the CA–CB complex. These studies provide novel structural insights to explain the role of the CA subunit in the mechanism of action of crotoxin. The crystal structure of the highly toxic and stable CA2CBb complex crystallized here allows us to identify key amino acid residues responsible for significant differences in the pharmacological activities of the two classes of crotoxin complexes. In particular, we show that critical residues Trp31 and Trp70 of the CBb subunit establish intermolecular polar contacts with Asp99 and Asp89, respectively, of the β-chain of CA2 and contribute to the stability and toxicity of the CA2CBb complex. These interactions also lead to decreased PLA2 activity by partially blocking substrate access to the catalytic dyad and by masking several interfacial binding surface residues important for PLA2 interaction with phospholipids.Identification of the binding interface between the CA subunits and the CB subunits of crotoxin is important for the structure-based design of antineurotoxic inhibitors. Since crotoxin displays numerous physiological functions, including antitumoral properties, knowledge of its three-dimensional structure will be useful for the understanding of these diverse effects.