Solution structure of an insect-specific neurotoxin from the new world scorpion Centruroides sculpturatus Ewing

We report the high-resolution solution structure of the 6.3 kDa neurotoxic protein CsE-v5 from the scorpion Centruroides sculpturatus Ewing (CsE, rang e southwestern U.S.). This protein is the second example of an Old World-li ke neurotoxin isolated from the venom of this New World scorpion. However, unlike CsE-V, which is the first Old World-like toxin isolated and shows bo th anti-insect and anti-mammal activity, CsE-v5 shows high specificity for insect sodium channels. Sequence-specific proton NMR assignments and distan ce and angle constraints were obtained from 600 MI-It 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed to p roduce a final family of 20 structures without constraint violations, along with an energy-minimized average structure. The protein structure is well- defined (0.66 and 0.97 D rmsd for backbone and all heavy atoms, respectivel y) with a compact hydrophobic core and several extending loops. A large hyd rophobic patch, containing four aromatic rings and other aliphatic residues , makes up a large area of one side of the protein. CsE-v5 shows secondary structural features characteristic of long-chain scorpion toxins: a two and a half-turn alpha -helix, a three-strand antiparallel beta -sheet, and fou r beta -turns. Among the proteins studied to date from the CsE venom, CsE-v 5 is the most compact protein with nearly 50% of the amide protons having l ong exchange lifetimes, but CsE-v5 is unusual in that it has loop structure s similar to both Old and New World toxins. Further, it also lacks prolines in its C-terminal 14 residues. It shows some important differences with re spect to CsE-V not only in its primary sequence, but also in its electrosta tic potential surface, especially around areas in register with residues 8, 9, 17, 18, 32, 43, and 57. The loss of anti-mammal activity in CsE-v5 and the differences in its anti-insect activity compared to that of other prote ins such as CsE-V, vl, and v3 from this New World scorpion may be related t o residue variations at these locations.