Functional Study Of Active Sites In Scorpion Insect Toxin BmK IT From Buthus Martensii Karsch

The scorpion venoms are a rich source of toxic peptides and have been shown to be composed of 28-76 amino acid residues. Mainly neurotoxic proteins or peptides can interact specifically with various ionic channels in excitable membranes, so they have been used as valuable probes for the elucidation of structure and function of ion channels. At the same time, scorpion neurotoxins are very useful models for studying the structure-function relationship of protein. BmK IT is an insect excitatory neurotoxin cloned in Buthus martensii Karsch which is a sort of scorpion mainly distributed in our country. This toxin is composed of 69 amino acids residues cross-linked by four disulfide bridges. In this study, the main residues in this toxin were characterized by in vitro studies.To date, of the excitatory toxins, only the 3D structures of Bj-xtrIT and BmK IT-AP have been determined by crystallographic studies, and the NMR solution structure of BmK-βIT was obtained. The result reveal that the functional surface ofβ-toxins is composed of two binding regions and a functional site:(i) a cluster of residues associated with the a-helix includinga putative'hot spot', referred to as the'pharmacophore' ofβ-toxins. (ii) A hydrophobic cluster, which is likely to confer the specificity, formed by a stretch of hydrophobic residues on the C-tail (for insect Na+ channels) or associated mainly with theβ2 andβ3 strands (for mammalian Na+ channels). (iii) A negatively charged residue (Glu-15) involved in voltage sensor trapping. Prediction of three dimensional structure of BmK IT was performed by SWISS-MODEL workspace. The 3D structure of BmK IT was similar to BmK IT-AP,Bj-xtrIT and BmKβIT. So site-directed mutagenesis was used to obtain the mutant expression plasmids:pTWIN1-rBmK IT(I25E)-His6, pTWIN1-rBmK IT (E15G)-His6, pTWIN1-rBmK IT C-terminal truncated-His6.The recombinant expression plasmid pTWIN1-BmK IT-His6 was constructed and the fusion protein DnaB- BmK IT-His6 was expressed in pTWIN1-BmK IT-His6/BL21(DE3). rBmK IT was eluted after intein self-cleavage on the Ni-NTA resin. The DnaB motif is biologically active since it can self-cleave efficiently at pH 6.5. The oxidative refolding treatment of rBmK IT was performed in the optimized condition (50 mM Tris-HCl, pH 8.5,2.0 mM GSH,0.5 mM GSSG). Intein self-cleaved efficiently when the pH was changed from 8.5 to 6.5. Circular dichroism spectrum analysis showed that rBmK IT, rBmK IT(I25E), rBmK IT(E15G) and rBmK IT C-terminal truncated contained a-helix and (3-pleated sheet structures.In the cytotoxicity assay, after adding MTT, the cell number in each well was calculated by the absorbance at 490 nm according to the linear response. Cells (104 cells/well) were added into wells containing various concentrations of rBmK IT, rBmK IT(I25E), rBmK IT(E15G) and rBmK IT C-terminal truncated. With the increasing concentration of rBmK IT, the number of Sf9 cells first increased(from 0μM to 10μM) and then decreased (from 10μM to 30μM). Different concentrations of the proteins corresponding the inflection points showed that rBmK IT(I25E)could increase the sensitivity of rBmK IT, but rBmK IT(E15G)could decrease the sensitivity of rBmK IT on Sf9 cells. The rBmK IT (C-terminal truncated) truncated C-terminal hydrophobic amino acids could cross the species boundaries and was effective on mammalian cells, for example, rat glioma C6 cells.In the electrophysiological assay (whole-cell patch-clamp recording) and far-western assay, the voltage-dependent sodium channel and the a subunit of sodium channel could not be detected in Sf9 cells, respectively. Then, we designed the experiment to co-locate tetrodotoxin (TTX) and rBmK IT or its mutants. TTX is an important and indispensable tool in neurobiology for binding the sodium channel specially. Sf9 and C6 cells were incubated with TTX and rBmK IT or its mutants. Then cells were stained with rabbit anti-TTX IgG followed by incubation with TRITC-conjugated anti-rabbit IgG. Then cells were stained with mouse anti-His6 antibody followed by incubation with goat anti-mouse IgG FITC. Colocalization results showed that the sodium channels existed on Sf9 cells and C6 cells membrane. The overlap between the location of rBmK IT, rBmK IT (I25E) or rBmK IT (E15G) and the TTX on the Sf9 cells membrane confirmed that rBmK IT, rBmK IT (I25E) and rBmK IT (E15G) could act on the sodium channel on Sf9 cells membrane. And the overlap between the location of rBmK IT (C-terminal truncated) and the TTX on the C6 cells membrane confirmed rBmK IT (C-terminal truncated) could act on the sodium channel on C6 cells membrane.Crystallization of rBmK IT was performed using the hanging-drop method.50 conditions (Crystal Screening Kit, Hampton Research) were used in order to get crystals. The results showed that pentagonal crystal could be obtained from a condition containing 0.1 M Sodium phosphate monobasic monohydrate,0.1 M Potassium phosphate monobasic,0.1 M MES monohydrate (pH 6.5) and 2.0 M Sodium chloride, at 28℃and the protein was dissolved in ddH2O.In this study, the mutants of rBmK IT were achieved by site-directed mutagenesis. Biological activity of rBmK IT and its mutants confirmed these amino acids or peptides played key roles in rBmK IT. The result laid a foundation for further structural and functional analysis of rBmK IT.