Structure–activity relationship of an α-toxin Bs-Tx28 from scorpion (Buthus sindicus) venom suggests a new α-toxin subfamily

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na+, K+, Ca+ or Cl−. An α-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3 ± 2Da), including eight cysteine residues, and shows very high sequence identity (82–94%) with other long-chain α-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJα-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na+ channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known α- and α-like toxins suggests the presence of a new and separate subfamily of scorpion α-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD50 0.088 and 14.3 μg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC50 = 0.01 μg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na+-channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical α-mammal (AaH-II), α-insect (Lqh-αIT), and α-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a “necklace-like” structure differing significantly in all α-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned α-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion α-mammal toxins.