NMR solution structure of BmK-βIT, an excitatory scorpion β-toxin without a ‘hot spot’ at the relevant position

BmK-βIT (previously named as Bm32-VI in the literature), an excitatory scorpion β-toxin, is purified from the venom of the Chinese scorpion Buthus martensii Karsch. It features a primary sequence typical of the excitatory anti-insect toxins: two contiguous Cys residues (Cys37-Cys38) and a shifted location of the fourth disulfide bridges (Cys38-Cys64), and demonstrates bioactivity characteristic of the excitatory β-toxins. However, it is noteworthy that BmK-βIT is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is the first example having a non-glutamate residue at the relevant position in the excitatory scorpion β-toxin subfamily. The 3D structure of BmK-βIT is determined with 2D NMR spectroscopy and molecular modeling. The solution structure of BmK-βIT is closely similar to those of BmK IT-AP and Bj-xtrIT, only distinct from the latter by lack of an α0-helix. The surface functional patch comparison with those of BmK IT-AP and Bj-xtrIT reveals their striking similarity in the spatial arrangement. These results infer that the functional surface of β-toxins is composed of two binding regions and a functional site. The main binding site is consisted of hydrophobic residues surrounding the α1-helix and its preceding loop, which is common to all β-type scorpion toxins affecting Na+ channels. The second binding site, which determines the specificity of the toxin, locates at the C-terminus for excitatory insect β-toxin, while rests at the β-sheet and its linking loop for anti-mammal toxins. The functional site involved in the voltage sensor-trapping model, which characterizes the function of all β-toxins, is the negatively charged residue Glu15.