Two-Dimensional ¹H Nuclear Magnetic Resonance Study of AaH IT, an Anti-Insect Toxin from the Scorpion Androctonus australis Hector. Sequential Resonance Assignments and Folding of the Polypeptide Chain

Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includes two and a half turns of α-helix running from residues 21 to 30 and a three-stranded antiparallel β-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the α-helix to an external strand of the β-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. These structure elements are very similar to the secondary structure reported in single crystals for either variant 3 from the scorpion Centruroides sculpturatus Ewing (CsE V₃) or toxin II from the scorpion A. australis Hector (AaH II). The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. This surface is made of an aromatic cluster that is surrounded by long hydrophobic side-chain residues, as well as the loops protruding out of it. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction.