Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic ß-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1-4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

Design of Crotoxin-Based Peptides with Potentiator Activity Targeting the ΔF508NBD1 Cystic Fibrosis Transmembrane Conductance Regulator.

We have previously shown that the CBb subunit of crotoxin, a ß-neurotoxin with phospholipase A2 (PLA2) activity, targets the human ΔF508CFTR chloride channel implicated in cystic fibrosis (CF). By direct binding to the nucleotide binding domain 1 (NBD1) of ΔF508CFTR, this neurotoxic PLA2 acts as a potentiator increasing chloride channel current and corrects the trafficking defect of misfolded ΔF508CFTR inside the cell. Here, for a therapeutics development of new anti-cystic fibrosis agents, we use a structure-based in silico approach to design peptides mimicking the CBb-ΔF508NBD1 interface. Combining biophysical and electrophysiological methods, we identify several peptides that interact with the ΔF508NBD1 domain and reveal their effects as potentiators on phosphorylated ΔF508CFTR. Moreover, protein-peptide interactions and electrophysiological studies allowed us to identify key residues of ΔF508NBD1 governing the interactions with the novel potentiators. The designed peptides bind to the same region as CBb phospholipase A2 on ΔF508NBD1 and potentiate chloride channel activity. Certain peptides also show an additive effect towards the clinically approved VX-770 potentiator. The identified CF therapeutics peptides represent a novel class of CFTR potentiators and illustrate a strategy leading to reproducing the effect of specific protein-protein interactions.