Abstract
Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKbeta1) belonging to the TsTXKbeta molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1-21 and 22-72 fragments (named N(1-21) and C(22-72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKbeta1) exhibited a low affinity for K(+) channels and cytolytic effects against bacteria and several eukaryotic cells. N(1-21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22-72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKbeta1 presents a typical scorpion toxin scaffold in water and its alpha-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1-21) and an ab initio structure model of MeuTXKbeta1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKbeta-related peptides and antiparasitic scorpines which both comprise the betaSPN (beta-KTxs and scorpines) family.