Collection Of Venom From Scorpion Buthus Sindicus Biology Essay

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The venom was collected from telson into a fine glass capillary. First the transparent pre venom was collected by gentle tapping of the telson for manual stimulation with iron electrode without current followed by electrical (direct current) stimulation of ~3 volts for the white, viscous post venom (Fig. 3.1). The pre- and post-venom were kept separately in lyophilized form at -20 °C till further use.

3.2. Characterization of pre- and post-venom from scorpion B. sindicus

3.2.1. SEC FPLC separation

Preliminary characterization of the pre- and post-venom was performed by size exclusion chromatography (Superdex 75 10/300GL) on Akta basic system (Pharmacia, Sweden) using 50mM ammonium acetate buffer pH 5.5. The elution profile of the pre and post venom samples revealed clear differences in the composition of the two venoms. In the pre-venom five peaks were observed as compared to 10 peaks in the post-venom (Fig. 3.2). Also the major peak of pre-venom contained low molecular mass peptides while the major peak of post-venom contained high molecular mass proteins followed by other peaks exactly superimposed with the peaks obtained in pre-venom but with low intensity. The pre-venom was chosen for further characterization in this study because of their small size, stability and medicinal significance.

3.2.2. RP HPLC separation of pre-venom

The single step purification of pre-venom was performed using RP-HPLC as described in material and methods. Approximately 41 peaks were collected manually as shown in highlighted peak area (Fig. 3.3). Peak 6, 8, and 28 were sequenced and reported earlier (Ali et al., 1998 and 2006). Peak 6 and 8 were synthesized and further characterized in this study. Peak 7 and 29 were also purified, sequenced, synthesized and characterized in this study (see following sections). The measured MH+ mass of native peak 7 was 3821.46 Da whereas that of peak 29 was 7273.3Da. These peaks have been named in accordance with their appearance and ascending retention time in RP-HPLC where the preface Bs stands for scorpion (Buthus sindicus) and Tx for the toxins (data is summarizes in Table I).

3.3. Characterization of scorpion short-chain neurotoxins (SCN)

3.3.1. Potassium channel inhibitor (Bs-KTx6)

3.3.1.1. Isolation and Bioactivity of native Bs-KTx6:

Peak number 6 (RT 32 min. - Fig. 3.3, Table I), designated as Bs-KTx6 (Bs for scorpion B. sindicus , K for potassium channel and Tx for toxin) was rechromatographed on RP-HPLC column (µRPC C2/C18) using gradient program D (Appendix I). The mass of this fraction obtained by MALDI-TOF MS was 4115.4Da (Fig. 3.4A-B). The preliminary screening of Bs-KTx6 toxin was performed on human voltage gated potassium channel (hVGKC) hKv1.1 which was expressed in CHO cells. Whole cell current traces in the absence and presence of Bs-KTx6 were obtained using different toxin concentrations. The data shown as normalized current represent mean ± S.E. of three experiments. The inhibitory concentration (IC50) of native Bs-KTx6 was found to be 1.3 ± 0.09µM (Fig. 3.4C).

3.3.1.2. Synthesis of Bs-KTx6:

Bs-KTx6 is a minor component of scorpion (B. sindicus) pre-venom. Realizing the low micromolar potencies Bs-KTx6 was synthesized by Fmoc solid phase peptide synthesis in order to establish its selectivity towards Kv1.x VGKCs. The crude linear and reduced peptide was purified on RP-HPLC column (µRPC C2/C18) using gradient program D (Appendix I). The purified peptide was allowed to fold into its native form in the presence of reduced/oxidized glutathione containing Guanidin HCl at pH7.6 for three days. After oxidation the peptide was desalted and purified as described in material and methods. The peptide mass determined by MALDI-TOF MS was 4120.9 (reduced form) and 4115.4 (oxidized form) shows a difference of ~6Da which nicely confirm the formation of three disulfide bonds. The chromatogram shows that the oxidized peptide elutes earlier as compared to the reduced peptide (Fig. 3.5). This reduction in the retention time of peptide after oxidation also confirms that the peptide has folded properly as compared to the linear reduced peptide. In order to get further evidence for proper folding the secondary structure of the synthetic peptide was determined by circular dichroism in the far UV range (190 - 250 nm). CD spectrum revealed a typical spectrum with dominating minima at 215 nm and maxima at 198 nm indicating the predominant β-sheet structure (Fig. 3.6).

3.3.1.3. Bioactivity of synthetic Bs-KTx6:

In order to establish the selectivity of Bs-KTx6, bioactivities of synthetic Bs-KTx6 were measured on subfamilies of voltage gated potassium channels (VGKCs) i.e., Kv1.x e.g., Kv1.1, Kv1.2 and Kv1.3. Figure 3.7 shows the normalized current inhibition by various concentrations of Bs-KTx6 on Kv1.1, Kv1.2 and Kv1.3 obtained by plotting the data as mean ± S.E. of at least three experiments. The IC50(s) calculated for the synthetic Bs-KTx6 were 0.78 ± 0.4µM (Kv1.1), 3.7 ± 1.3µM (Kv1.2) and 7.7 ± 1.7pM (Kv1.1). The current traces in presence and absence of toxin Bs-KTx6 concentration 100nM (Kv1.1), 10µM (Kv1.2) and 10pM (Kv1.3) have also been shown for clarity. Bs-KTx6 is thus finally characterized as a potent blocker of Kv1.3 and belongs to the scorpion short chain neurotoxin family αKTx3. The IC50 and/or EC50 of the members of αKTx3 family have been compared with the IC50 of Bs-KTx6 determined in this study (Table II). Comparison revealed that Bs-KTx6 is the second most potent as well as selective blocker of Kv1.3 in αKTx3 family in its natural and native form after AgTx2 from scorpion (Leiurus quinquestriatus Hebraeus). Comparison of the IC50 of different αKTx3 mutants also shows that "ADWX-1" (a triple-mutant of BmKTx from scorpion Buthus martensii Karsch) is one of the most potent and selective peptidyl blocker of Kv1.3 developed so far.

3.3.1.4. In silico studies of Bs-KTx6

3.3.1.4.1. Sequence analysis and search for best template for modelling

Multiple sequence alignment of Bs-KTx6 with other known members of "α-KTx3 subfamily" of scorpion short-chain neurotoxins (SCN) active against K+-channel (Kv1.x) was established (Fig 3.8). Sequences are aligned according to their conserved cysteine residues (bold letters) and known 3D structures (marked by #) using the program ClustalX. Pairing of the conserved disulfide bonds is indicated on top and positions are marked by numbers. Residual position from 1-38 are marked according to the sequence of Bs-KTx6. Global electrostatic charges (GEC: R/K = +1; H = +0.5 and E/D = -1) and % identities of the toxins related to Bs-KTx6 are listed at the right side of the sequences. Consensus amino acid residues (e.g. Arg24, Phe25 & Lys27) involved in interacting surface of the a-KTx3 with Kv1.x are also marked. Phylogenic tree of a-KTx3 toxins derived from these primary sequences using the neighbor joining distances method within the Phylip package is also shown in figure 3.9. The overall homology between the different members of α-KTx3 is quite high i.e. 70 - 91%, suggesting the obvious homogeneity in their target i.e., Kv1.x VGKCs. The toxin OdK2 recently isolated from Iranian scorpion (Odonthobuthus doriae) has the highest sequence homology (91%) with Bs-KTx6 but its 3D structure has not been established yet. AgTx2 from Leiurus quinquestriatus herbraeus (Israelian yellow scorpion) was found to have 86% sequence identity with Bs-KTx6. Fortunately the 3D NMR structure of AgTx2 is available in protein data bank (pdb i.d. 1AGT). Thus the 3D predicted structural model of Bs-KTx6 was established using AgTx2 as template by the Linux based program Modeller 6v2 and the on-line SWISS-MODEL server.

The three-dimensional homology model structure of Bs-KTx6 revealed a typical and highly conserved structural scaffold common to all scorpion SCNs (Fig. 3.10A) i.e. an a-helix (red), three-anti-parallel b-sheets (cyan) connected by variable loops (silver) stabilized by three intra-chain disulfide linkages (shown in yellow ball and sticks). Structural superposition of Bs-KTx6 with all other known members of the subfamily a-KTx3 active against Kv1.x family of VGKCs (Bs-KTx6, blue; AgTx2, red; OsK1, cyan; BmKTx1, gray; KTx, black; BoiKTx1, green; Odk2, meganta) shows root mean square (RMS) deviation of less than 1Š(Fig.3.10A and B). Despite the very high sequence identity and conserved 3D structures among the members of α-KTx3 family, subtle differences in residual change, side chain confirmations (Fig. 3.10C to H) and over all global electrostatic charge distribution (Fig. 3.11) were found to be crucial in determining the channel blocking activity and selectivity towards different VGKCs.

3.3.1.4.2. Sequence alignment and modelling of Kv1.3 channel

Multiple sequence alignment of the pore region of human potassium channel Kv1.x family with Shaker and one of the four subunits of bacterial K+-channel (KcsA) using the program CustalX has been shown in table III. Location of amino acid residues of various K+-channels where the mutations influence the binding activity and/or reported to be involved in the interaction with different scorpion toxins are also marked with asterisks. As Bs-KTx6 has been found to be potent and selective at mKv1.3 in the electrophysiological experiment, a model of mKv1.3 was constructed in silico by replacing pore region (Ala50 to Thr85) of bacterial potassium channel, KcsA (pdb i.d. 1BL8) with pore region of mKv1.3 (Ala372 to Thr407). mKv1.3 shares 52.7% sequence identity with pore region of bacterial potassium channel KcsA. The backbone root-mean-square deviation of the P-loop region of mKv1.3 after superimposition on the template KcsA revealed a RMSD of 0.2Å. Overall sequence identity of the Kv1.x family was <20%, while the pore region showed 50% sequence identity (Table III) suggesting the importance and similar role of pore region of ion conductance in all members of the VGKCs family. It is also worth mentioning here that the sequence of hKv1.3 pore region is identical with rabbit and panda (100%) but variable with chicken (94%; T436S & V464I), rat (94%; T375S and S379N) and mouse (94%; T378S and S382N).

3.3.1.4.3. Docking studies and interaction surfaces

Figure 3.12 shows interaction complex between Bs-KTx6 and Kv1.3 as established by soft molecular docking studies. Cluster of toxin Bs-KTx6, obtained on the basis of electrostatic charge was selected using first 15 best solutions out of 5000. The Lys27 chain is opened in all solutions which is clearly occluding the channel pore. The residues of Bs-KTx6-Kv1.3 complex (shown in line representations) are equally important for AgTx2 interaction with Kv1.3. The toxin Bs-KTx6 has been translated vertically to annotate the number and give visualization access to important residues constructing the interaction surfaces. Potassium ions are shown in purple color. Channel is also illustrated in terms of interacting electrostatic surface potential with added annotation of the side chains of important variable residues of the turret and guard sequence (red color). The position of two mutations i.e. T378S and S382N between human and mouse Kv1.3 is also depicted in bold case. Bs-KTx6 backbone is shown in blue color and in order to reveal the "plug-in" respective position of the pore occluding side chain of Lys27 is also annotated.

3.3.2. Characterization of CFTR inhibitor-like peptide (Bs-Tx7)

3.3.2.1. Isolation and purification

The fraction collected as Bs-Tx7 (Fig. 3.3) was further rechromatographed on C2/C18 column using gradient program E (Fig. 3.13). The SDS PAGE gel (15%) (insert figure 3.13) confirmed the purity of the peptide Bs-Tx7. The exact mass was established by MALDI-TOF MS 3821.47 Da (Fig. 3.14).

3.3.2.2. Peptide digestion and sequencing

Primary structure of the purified Bs-Tx7 was established by a single one goal automated Edman sequencing (yield >95%) but realizing the very different pairing of disulfide bonds, the native peptide was also subjected to enzymatic cleavage for 24 hrs at 37°C. Off-line separation of the tryptic digest was performed using C12 RP-HPLC (Fig. 3.15). All major peaks were collected, dried and subject to purity check by mass spectroscopy followed by Edman sequencing. Three peaks (i.e. T1, T2 and T3) were identified as specific tryptic cleavage products (oxidized) and two peaks named as T3a and T3b were found to be the results of non specific cleavage (Table IV). The masses of the peptide, determined by ESI-MS, revealed very good agreement between the calculated and measured masses (Table IV). Thus, the complete primary structure was successfully established by overlapping the sequences of tryptic fragments with the intact native peptide. The position of disulfide bonds were confirmed using established techniques.

3.3.2.3. Synthesis of Bs-Tx7

The purification of the synthetic peptide toxin Bs-Tx7 has been shown in figure 3.16. The toxin was synthesized in reduced or linear form (blue peak) and then oxidized as described earlier. Oxidation of cysteine residues allows the peptide to form disulfide linkage necessary for proper folding to become active against its target like native toxin. The fully active and oxidized peptide (red peak) was eluted earlier then the reduced peptide indicating that the toxin has acquired proper folds. The average molecular masses of the toxins obtained in reduced and oxidized form shows the difference of ~8Da indicating the presence of 8 cysteines and possibly 4 disulfide linkages in the native peptide. Unfortunately, the yield of the synthetic Bs-Tx7 was not sufficient to perform CD analysis but the peptide exhibits activity which is a good indicator besides mass and retention time that the peptide folds perfectly.

3.3.2.4. In silico studies of Bs-Tx7

3.3.2.4.1. Sequence analysis and homology modeling

Multiple sequence alignment of Bs-Tx7 was performed with its homologues obtained from Fasta/Blast searches and the literature review. Alignment was based on their conserved cysteine residues (bold letters highlighted in grey color) and known 3D structure of related toxins (marked by #) using the program ClustalX. The conserved disulfide bond pairing is indicated on top and positions are marked by numbers. Residual positions are marked according to the sequence of Bs-Tx7 (Fig. 3.17). Beside cystine residues, other conserved residues at N- and C- terminals have also been highlighted in yellow color. Global electrostatic charges (GEC: R/K = +1; H = +0.5 and E/D = -1) and % identities of the toxins related to Bs-Tx7 are listed at the right side of the sequences. Phylogenic tree derived from the primary sequences using the neighbor joining distances method within the Phylip package is also shown in figure 3.18. Bs-Tx7 shares variable (49 - 88%) sequence identity with other chlorotoxin-like, CFTR inhibitor-like and insectotoxin-like peptides. Bs-Tx7 revealed highest 88% sequence identity with Bs14 and 82% identity with CFTR inhibitor GaTx1 toxin isolated from scorpion Leiurus. quinquestriatus Hebraeus. Unfortunately, the 3D structural details of GaTx1 is not yet established but the structure of some other members such as chlorotoxin (66%), insecto-toxins Lqh8/6 (60%) and BeI5A (71%) have been resolved by NMR spectroscopy (Fig. 3.17). Thus, the 3D structural models of Bs-Tx7, Bs-Tx14 and GaTx1 and a chlorotoxin-like peptide Bs-Tx8 have been established using the known coordinates of chlorotoxin and rigorously utilized for structural comparisons.

The three-dimensional homology model structure of Bs-Tx7, BsTx8, Bs-Tx14 and GaTx1 revealed a typical and conserved structural scaffold or motif common to all scorpion SCNs (Fig. 3.19) i.e., a short a-helix (red), two or three-antiparallel b-sheets (cyan) connected by variable loops (silver). But in contrast to -KTx3 family, these are stabilized by four intra-chain disulfide linkages (shown in yellow ball and sticks). Structural superposition of Bs-Tx7 with all other known and modeled members of the toxins active against CLC/CFTR channels (Bs-Tx7, black; CTX, red; Bs-Tx8, blue; GaTx1, green and Bs-Tx14, meganta) shows root mean square (RMS) deviation of less than 1Š(Fig. 3.20). Despite high sequence identity and conserved 3D structures among the members of CLC/CFTR inhibitor family, subtle differences in terms of residual change, side chain confirmations (Fig. 3.20A to E) and over all surface complementarities and electrostatic charge distribution (Fig. 3.21) have been observed and seems crucial in channel blocking activity and selectivity.

3.3.3. Characterization of MMP2 modulators

3.3.3.1. Synthesis of MMP2 FRET-substrate

A fluorescence resonance energy transfer based substrate for hMMP2 was synthesized. The peptide has seven amino acids residues i.e., Pro-Leu-Gly-Leu-Lys-Ala-Arg-NH2 with a fluorophore 7-methoxycoumarin-4-yl-acetyl (Mca) attached with N-terminal proline and a quencher 2,4-dinitrophenyl (Dnp) attached with lysine while the scissile peptide bond is between glycine and leucine. The substrate was purified using RP HPLC (Fig.3. 22) as described in material and methods. Substrate after cleavage produces two fragments separating the fluorophore and quencher and the difference in the fluorescence was monitored at 340nm excitation and 405nm emission wave length respectively.

3.3.3.2. Kinetics of FRET substrate

The Lineweaver-Burk plot (Fig. 3.23) has been plotted between different concentrations of newly synthesized FRET substrate and the hMMP2. The kinetic parameters Km and Vmax obtained using calculation on microsoft excel program have been found to be 13.6µM and 902.9 µM/min respectively.

3.3.3.3. MMP2 inhibitors

3.3.3.3.1. Synthesis of Chlorotoxin (CHL)

In order to validate our FRET based MMP2 enzyme assay, a known inhibitor of hMMP2 e.g., Chlorotoxin (CHL) was also synthesized as positive control. Figure 3.24 shows the RP-HPLC chromatograms superposition of purified reduced (blue) and oxidized (red) chlorotoxin. The retention time for reduced and oxidized chlorotoxin measured was 23 and 27min, respectively, and the difference in their measured masses by MALDI-TOF-MS (~8Da) are in good agreement for the presence of eight cysteine residues and four disulfide linkages.

3.3.3.3.2. Bioactivity of Chlorotoxin

Bioactivity of the synthetic chlorotoxin was analyzed using synthetic FRET based peptide substrate of MMP2. Despite low yield in synthesis, Chlorotoxin inhibits the activity of hMMP2 with IC50 0.09µM (Fig. 3. 25A) which nicely support the perfect folding of the synthetic chlorotoxin. The inhibitory activity of chlorotoxin was also complemented by zymographic determination using 0.1% gelatin as substrate (Fig. 3.25B). Negative staining of the gel shows white band of catalysis (~68 kDa) in the lane 1 from the right representing active enzyme MMP2 (0.002 µM) as standard while, lane 2 shows the same amount of enzyme in presence of chlorotoxin (0.625 mM). Result shows around 50% of enzyme inhibition under described zymographic conditions.

3.3.3.3.3. Docking studies

To test the hypothesis, whether or not the chlorotoxin really inhibits chloride channel in silico? 3D structure of chlorotoxin (pdb i.d. 1CHL) and its possible target chloride channel (for e.g. from E.coli; pdb i.d. 1KPK) were obtained from protein data bank (Fig. 3.26). 5000 docking solutions were obtained using BiGGER protein-protein soft docking software. All 5000 solutions were analyzed and the interaction of chlorotoxin in all dimensions especially extra-cellular and intracellular regions were accumulated and plotted in a graph shown in figure 3. 27. Although it is very difficult to rationalize the possible interaction between the CLC and chlorotoxin, the data suggests that the chlorotoxin binds maximally on intracellular sites.

3.3.3.3.4. Chlorotoxin-like peptide (Bs-Tx8)

The primary structure of a chlorotoxin-like peptide Bs-Tx8 was also reported earlier by Ali and coworkers (1998) from the scorpion B. sindicus venom. As Bs-Tx8 is a natural and closest homologue of chlorotoxin (80%), we have also synthesized Bs-Tx8 in this study as described in material and method and results are presented in Figure 3. 28. A difference in retention time of ~1min was observed between the purified reduced (blue) and oxidized peptide (red). Different concentrations of Bs-Tx8 when incubated at 37°C for 1hr with hMMP2 showed inhibition of the enzyme. The FRET substrate for hMMP2 synthesized in this study was used to calculate the IC50 of chlortoxin-like peptide Bs-Tx8. The observed IC50 was 6.5µM (Fig. 3.29A) which revealed far less potencies then synthetic chlorotoxin inhibition (about 72 folds) as described under similar experimental conditions. Variation in inhibition potencies may nicely be correlated with the few but drastic sequence variation such as Arg1→Met, Lys3→Met, Prol10→His, Ser13→Ala, Ala17→Asp, Lys25→Arg and a C-terminal deletion in Bs-Tx8 (see Fig. 3.17 for details).

3.3.3.3.5. MMP2 activation by Bs-Tx7

The Bs-Tx7 which was described above as CFTR inhibitor-like peptide on the basis of close sequence homology with GaTx1 from Leiurus quinquestriatus Hebraeus (82%) was also tested for hMMP2 inhibiting activity. In contrast to chlorotoxin and Bs-Tx8, which inhibits MMP2 activities, different concentrations of Bs-Tx7 were found to increase the activity of hMMP2 under identical assay conditions (Fig. 3.29B). Although we have not performed the CFTR inhibition activities, the lack of inhibition of hMMP2 and very high sequence homology with GaTx1 suggest that CFTR is probably the cellular target of Bs-Tx7.

3.3.4. Characterization of scorpion long-chain neurotoxin (LCNs)

3.3.4.1. Sodium channel modulator

3.3.4.1.1. Isolation and purification

Peak number Bs29 (RT 47.5min, Fig. 3.3) was rechromatographed on RP HPLC column using the conditions as described in material and methods (Appendix I). The molecular weight of Bs-Tx29 determined by mass spectrometry using MALDI-TOF MS was found to be 7273.3 ± 2 Da (Fig. 3.30).

3.3.4.1.2. Peptide sequencing

The primary structure of the purified Bs-Tx29 was determined by direct automated Edman degradation analysis. The repetitive yield in each cycle during the N-terminal sequencing was >95%. The average molecular mass calculated from the established complete sequence (7272.2 Da), is consistent with the mass measured by MALDI-TOF-MS ([M + H]+ = 7273.3 ± 0.2 Da). Thus, the complete amino acid sequence of Bs-Tx29 has been established as single polypeptide chain composed of 65 amino acid residues including eight half-cystine residues (Fig. 3.31A-B). Primary structure of Bs-Tx29 was found to be isotype of Bs-Tx28 (90%), a potent modulator of TTX-sensitive VGSC recently reported by us.

3.3.4.1.3. Extracellular electrophysiology

Figure 3.32 presents the Bs-Tx29-induced afferent nerve response measurement in presence and absence of tetrodotoxin (TTX). Bs-Tx29 diluted in Kreb's solution (0.002µg/ml) was applied as control (left-hand panels) and the same concentration of Bs-Tx29 with TTX was also tested (right-hand panel). Bs-Tx29 induced totally eliminated afferent nerve response. The luminal pressure (LP) recording shown in the lower panel reflects the gut motility. After application of Bs-Tx29, a slight inhibition in gut spontaneous contractile activity was observed accompanied by the activation of nerve discharge. In our presented data, baseline discharge was determined during a 5 min period prior to injection. Increase in mesenteric discharge above the baseline following injections were expressed as impulses per second (area under curve AUC imp/s) and presented as means ± standard error of the mean (SEM). Dose-dependent nerve responses of the Bs-Tx29 (n = 4) was found to be identical with Bs-Tx28 with the EC50 0.01µg/ml (right-hand panel), suggesting that two toxins exclusively bind with the TTX-sensitive voltage gated sodium channels (VGSCs).

3.3.4.1.4. Comparative CD analysis of Bs-Tx28/29

A comparative far-UV CD spectrum of the two toxins has been presented in figure 3.33. Bs-Tx29 shows intense minima at 209 nm and mild positive maximum at 198nm in the CD spectrum suggesting the presence of predominant β-sheet structure. A curve in the minima in the region of 220 to 225 nm indicates the contribution of α-helix. In addition to this shows Bs-Tx28 (closest homologue of Bs-Tx29) shows minor Cotton effects at 235 nm like many other scorpion toxins which is absent in Bs-Tx29. This feature may be nicely correlated with major mutations of Phe40 → Leu and Trp43 → Tyr in the core domain in Bs-Tx28 besides effect of other mutations and an additional residue at the C-terminus in Bs-Tx29. On the other hand, temperature variable CD spectra in the far-UV region revealed quite a high thermal stability for both toxins i.e. up to 90°C, accompanied by a small reduction of the intensity of the negative Cotton effect around 215 nm (insets in figure 3.33). While in the presence of β-mercaptoethanol or DTT (as reducing agent) at 90°C, the thermal stability of the two toxins was totally lost, suggesting that the stability rely mainly on the highly conserved disulfide linkages.

3.3.4.2. In silico studies of Bs-Tx28/29

3.3.4.2.1. Sequence and phylogenic analysis

For comparison, sequence homology search of the established sequence of Bs-Tx29 in different gene, protein, and SCORPION databases was performed. A multiple sequence alignment and the phylogenic tree of Bs-Tx29 with all other known scorpion α-toxins is presented in figure.3.31A-B. Comparing Bs-Tx29 with other α-toxins, a striking degree of sequence identity (82-94%) is observed with previously suggested α-mammal toxins (e.g., Lqq-IV and Lqh-IV from scorpion Leiurus sp.), α -insect toxins (e.g., BJα-IT (from Judean black scorpion Buthotus judaicus), and OdK-1 (from Indian yellow scorpion O. doriae venom), which are known to interact with site-3 of VGSCs. Taking some recent sequence data in account, such as A9 (Androctonus bicolor), AamH2 (Androctonus australis Hector) and AmmVIIIrgp1-3 (Androctonus mauretanicus mauretanicus), phylogenetic analysis clearly presents this cluster of toxins as new subfamily of α-neurotoxins (Fig. 3.31B). The residual differences between Bs-Tx28 and 29 (includes Glu33→Asp, Phe40→Leu, Trp43→Tyr, Gly46→Ala, Asp54→Val, Lys55→Glu and a C-terminal extension in Bs-Tx29 i.e. Ala66) which was observed in the N-terminal, RT-CT- and core domains were also highlighted by red color in sequence alignment as well as their position in their respective 3D structures (Fig. 3.34).

3.3.4.2.2. Homology modeling and comparative 3D structure analysis

In order to gain 3D structural insight, homology model structures of Bs-Tx28, Bs-Tx29 and Bjα-IT were established using the known 3D coordinates of -mammal specific AaH-II (pdb id 1ptx), -insect selective Lqh-IT (pdb id 1lqh & 2asc), and -like toxins BmK-M1 and BmK-4 (pdb id 1sn1 & 1sn4). Structures illustrate the typical folding pattern of secondary structural elements (Fig. 3.34) i.e., an α-helix (red) and three-stranded anti-parallel β-sheets (cyan) connected by variable loops (silver) and stabilized by four conserved intra-chain disulfide linkages (C13-C64, C17-C37, C23-C47, and C27-C49; yellow sticks). Assessment of the reliability of the predicted models checked by PROCHECK and PROSA revealed satisfactory stereochemistry and overall energy statistics. Moreover, variability among the models and the known template structures (for e.g., α-mammal AaH-II from scorpion Androctonus australis Hector, α-insect Lqh-αIT from scorpion Leiurus quinquestriatus Hebraeus, and α-like BmK-M1 and 4 from scorpion Buthus martensii Karsch were determined by the superposition of Cα-traces and the backbone atoms revealed RMSD values <1Š(Fig. 3.34).

As expected subtle variations have been observed within the proposed structural and functional domains (i.e., Nt-, Core-, and RT-CT-domains) which construct a ''necklace-like structure'' in all scorpion α-toxins. Comparative 3D structural profiles of Bs-Tx29 with other representatives of each subfamily of scorpion α-toxins are shown in figure 3.35A (termed as phase A). All amino acid residues constituting the above mentioned three functional domains were found to assembled in a necklace-like structure (or a belt of amino acids) surrounding the toxin molecules. On the other hand, comparing the phase B for each α-toxin subfamily (Fig. 3.35B) revealed a conserved hydrophobic surface comprising of seven aromatic amino acid residues which contribute to both structural and functional roles. The differences in this phase remain associated within the same three functional domains which constitute the killer-necklace. A comparative 3D structural insight of the variable RT-CT domain (i.e. the protruding pendant in the killer-necklace) of different α-toxins has also been constructed and presented in figure 3.36. Key distinguishing features identified in this comparison includes (1) the presence of a non-proline cis/trans peptide bond between residue 9/10 and 10/11 (variable sequence) which is precisely regulated by (2) a molecular switch present at position 8 (Lys/Asp/Ala), and (3) stabilized by a complex hydrogen-bonding pattern (mainly contributed by the conserved N12/13 and the variable C-tail) and a conserved fourth disulfide bond.

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