Molecular Diversity and Functional Evolution of Scorpion Potassium Channel Toxins

Summary Scorpion toxins affecting K + channels (KTxs) represent important pharmacological tools and potential drug candidates. Here, we report molecular characterization of seven new KTxs in the scorpion Mesobuthus eupeus by cDNA cloning combined with biochemical approaches. Comparative modeling supports that all these KTxs share a conserved cysteine-stabilized α -helix/ β -sheet (CS αβ ) structural motif despite the differences in protein sequence and size. We investigated functional diversification of two orthologous α -KTxs (MeuTXK α 1 from M. eupeus and BmP01 from M. martensii ) by comparing their K + channel-blocking activities. Pharmacologically, MeuTXK α 1 selectively blocked Kv1.3 channel with nanomolar affinity (IC 50 , 2.36 ± 0.9 nM) whereas only 35% Kv1.1 currents were inhibited at 3 µM concentration, showing more than 1271-fold selectivity for Kv1.3 over Kv1.1. This peptide displayed weak effect on Drosophila Shaker channel and no activity on Kv1.2, Kv1.4, Kv1.5, Kv1.6 and hERG channels. Although BmB01 and MeuTXK α 1 have similar channel spectrum, their affinity and selectivity for these channels largely varies. In comparison with MeuTXK α 1, BmP01 only exhibits a sub-micromolar affinity (IC 50 , 133.72 ± 10.98 nM) for Kv1.3, showing a 57-fold less activity than MeuTXK α 1. Moreover, it lacks ability to distinguish between Kv1.1 and Kv1.3. We also found that MeuTXK α 1 inhibited the proliferation of activated T cells induced by phorbol myristate acetate (PMA) and ionomycin at micromolar concentrations. Our results demonstrate that accelerated evolution drives affinity variations of orthologous α -KTxs on Kv channels and indicate that MeuTXK α 1 is a promising candidate to develop an immune modulation agent for human autoimmune diseases.

In this work, we report seven new KTx genes expressed in the scorpion M. eupeus venom gland and their relationship with other known toxins based on sequence, structural and evolutionary analysis. Experimental data is provided to support functional diversification between two orthologous toxins through accelerated amino acid substitutions. We found that MeuTXKα1, an orthologue of the M. martensii toxin -BmP01, has properties that make it an attractive candidate for development of an immune modulation agent for human autoimmune diseases with therapeutic potential, which include 1) High-affinity on Kv1.3 (IC 50 , 2.36 ± 0.9 nM); 2) More than 1271-fold selectivity for Kv1.3 over Kv1.1; 3) Lacking activity on Kv1.2, 1.4, 1.5, 1.6, and hERG channels; 4) Inhibition on the proliferation of activated T cells by PMA and ionomycin.

Construction and screening of cDNA library
The construction of cDNA library from the M. eupeus venom gland has been described previously (12). Clones carrying an insert of 300 -1,000 bp potentially encoding venom peptide precursors were selected for DNA sequencing by primer T25V. Nucleotide sequences MeuTXKβ5-NHD(S) that was predicted by an ab initio modeling method on the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) due to the lack of a suitable template for its extended N-terminus. In the comparative modeling, aligned sequences of target and template were applied to build models through the "Alignment Mode" option, and the model quality was evaluated by Verify3D. Structural superimposition was performed at MultiProt (http://bioinfo3d.cs.tau.ac.il/MultiProt/) to identify a conserved functional motif.
MOLMOL (molmol-2k.2.0) (13) was used to display, analyze and manipulate toxin structures, in which electrostatic potentials mapped on the model structure surface were calculated by the "simplecharge" command and blue and red surface areas indicate positive and negative charges, respectively.

Isolation and purification of MeuTXKα1 and BmP01
Purification approaches used here have been described previously (14). Briefly, M. by guest on May 8, 2020 6 eupeus or M. martensii (previously called Buthus martensii (15)) crude venoms collected by an electrical stimulation method was resuspended in 0.1% trifluoroacetic acid (TFA, v/v) and directly subjected to RP-HPLC isolation. The Agilent Zorbax 300SB-C18 (4.6x150 mm, 5 µm) was equilibrated with 0.1% TFA in water (v/v) and peptide components were eluted from the column with a linear gradient from 0 to 60% acetonitrile in 0.1% TFA in water (v/v) within 60 minutes with a flow rate of 1ml/min. The UV absorbance trace was followed at 225 nm. All well-defined peaks were separately collected and rerun on the same column to purify these peptides further. Purity of MeuTXKα1 and BmP01 was identified by MALDI-TOF and Edman degradation which determines their N-terminal sequences. The amino acid sequence of MeuTXKα1 has been deposited in the UniProtKB protein database (http://www.ebi.ac.uk/uniprot/) under the accession number of P86400.

Electrophysiological recordings
Two-electrode voltage-clamp recordings were performed at room temperature (18-22°C) by guest on May 8, 2020 7 using a Geneclamp 500 amplifier (Axon Instruments, USA) controlled by a pCLAMP data acquisition system (Axon Instruments, USA). Whole cell currents from oocytes were recorded 4-5 days after injection. Bath solution composition was (in mM): NaCl, 96; KCl, 2; CaCl 2 , 1.8; MgCl 2 , 2 and HEPES, 5 (pH 7.4). Voltage and current electrodes were filled with 3M KCl. Resistances of both electrodes were kept as low as possible (<1.0 MΩ). The elicited currents were filtered at 1 kHz and sampled at 2 kHz using a four-pole low-pass Bessel filter.
Leak subtraction was performed using a P/4 protocol. Kv1.1-Kv1. 6 and Shaker currents were evoked by 500 ms depolarizations to 0 mV followed by a 500 ms pulse to -50 mV, from a holding potential of -90 mV. Current traces of hERG channels were elicited by applying a +40 mV prepuls for 2 s followed by a step to -120 mV for 2 s. To assess the concentration dependency of the MeuTXKα1 induced inhibitory effects, dose-response curves were constructed. The percentage blocked current was plotted as a function of increasing toxin concentrations. Each experiment was performed at least 3 times (n ≥ 3). All data are presented as mean ± standard error of the mean.

Proliferation assay of T-cells on PMA/Ionomycin
C57BL/6 mice (6-week-old, male) were purchased from Beijing Laboratory Animal Research Center (Beijing, China). All mice were maintained in specific pathogen-free facility and were housed in microisolator cages containing sterilized feed, autoclaved bedding, and water. Single-cell suspensions were prepared by grinding the spleen tissues with the plunger of a 5-ml disposable syringe and were then suspended in RPMI1640 medium. Splenocytes were treated with a hemolytic buffer (17 mM Tris-HCl and 140 mM NH 4 Cl, pH 7.2) to remove red blood cells as described before (17).

Isolation and characterization of M. eupeus K + channel toxin transcripts
From the cDNA library prepared from M. eupeus venom glands, we isolated and identified new transcripts encoding precursors of seven KTx-like peptides (Fig. 1A).
According to their sequence and structural features, we named these peptides MeuTXKα1, MeuTXKα2, MeuTXKα3, MeuTXKα4, MeuTXKβ3, MeuTXKβ4, and MeuTXKβ5. Of them, 9 cysteines with similar alignment pattern to known KTxs (8), indicating they may adopt a typical CSαβ folding. Structural analysis revealed that several indel mutations in Mesobuthus K + channel toxins are primarily located in three loops (Fig. 1B). Overall, most of these new toxin-like peptides are cationic due to the presence of 1.7 to 7.7 net positive charges.

New members of the α-KTx8 subfamily
The α-KTx8 subfamily is composed of five highly similar members (α-8.1-α-8.5), including AmP01, BmP01, LpII, LpIII, and OdK-1 ( Fig. 2A and B) (19)(20)(21)(22). MeuTXKα1  (19). These two peptides differ from other subfamily members by at least five amino acids, of which three are located on the turn linking the α-helix and the first β-strand, a key region characterized to be important in interacting with Kv channels. There is a lysine at position 18 that is conserved across the subfamily ( Fig. 2A). Such a lysine has been thought to be the most crucial amino acid for Kv channel blockade in many α-KTxs (8) and in some cases a hydrophobic moiety (normally Phe or Tyr) at a distance of approximately 6-7 Å is also needed to form a functional dyad (9).
Comparative modeling confirms that the overall fold of MeuTXKα1 is very similar to that of BmP01, which is composed of an α-helical region spanning residues 3 to 12 and two-strands of β-sheet spanning residues 16-19 and 24-27. The electrostatic potential of MeuTXKα1, calculated by MOLMOL, was characterized by a large negative zone around E 4 , D 5 , E 8 and D 22 and a small positively charged zone composed of K 13 and K 23 (Fig. 2C).
by guest on May 8, 2020

MeuTXKα3: a novel toxin-like peptide with a typical dyad motif and a cationic surface
The precursor of MeuTXKα3 contains 60 amino acids, including an N-terminal signal peptide of 22 residues, a mature peptide of 37 residues, and an extra C-terminal Gly that could be removed in post-translational processing to form an amidated peptide, as observed in two bee toxins -apamin and mellitin (23,24). MeuTXKα3 is a novel toxin-like peptide with very low sequence similarity to KTxs characterized so far ( Fig. 3A and B). However, this peptide has typical structural residues for the formation of CSαβ folding, which include six cysteines and one glycine in the GKC motif (7).
Structural model of MeuTXKα3 provides evidence supporting its possible K + channel-blocking function: 1) As predicted from its +7.7 net charges, this molecule possesses a rather large positively charged molecular surface around R 12 , K 13 , R 16 , R 25 , K 27 , and R 34 . On the opposed surface of this molecule, there is a small positively charged zone composed of three lysines at sites 17, 21 and 22 (Fig. 3C); 2) A dyad comprising K 27 and F 36 can be well superimposed with that of charybdotoxin (ChTX), a well-characterized scorpion α-KTx isolated from the venom of Leiurus quinquestriatus [7], at an ideal distance of 6.32 Ǻ between the lysine Cα atom to the center of the aromatic ring of F 36 (Fig. 3D).

MeuTXKα4: a novel toxin-like peptide having a double cysteine in its N-terminus
The precursor of MeuTXKα4 is composed of 63 residues with an N-terminal signal peptide of 28 amino acids that shares 64% similarity to that of BmK86 (25), a newly characterized toxin targeting Kv1.3 from M. martensii (Fig. 4A). Overall, mature MeuTXKα4 represents a novel peptide with low sequence similarity to several toxins from the α-KTx3 subfamily and BmK86 ( Fig. 4B and C), however, it has six cysteines with similar alignment pattern to other known KTxs, which could make it fold into a CSαβ structure, as confirmed by comparative modeling (Fig. 4D). Electrostatic potential analysis demonstrates that this peptide has a large positive zone around R 21 , K 23 and R 29 (Fig. 4D).

BmTXKβ-related peptides
MeuTXKβ3-MeuTXKβ5 are three highly similar peptides with 30-80% sequence identity to BmTXKβ and related toxins (Fig. 5A and B). After the signal peptide is removed, a mature peptide of 66-68 residues can be released. Considering their high-degree of sequence similarity to TcoKIK (10), we hypothesized that these peptide precursors may also have additional processing pattern to remove an N-terminal 19 residues after the signal peptide.
Due to no suitable template to build the full-length structures of these molecules by comparative modeling, computational ab inito predition was chosen as an alternative, which suggests that these peptides adopt a two-domain architecture, as previously proposed in the βSPN family of scorpion venom-derived antimicrobial peptides (AMPs) (14,26), in which the N-terminal part is cysteine-free and can form an α-helical conformation whereas the C-terminal part is a typical CSαβ fold (Fig. 5C), consistent with the model structure of the C-terminal part obtained by comparative modeling based on the scyllatoxin structure (PDB code 1SCY) (Fig. 5D). Interestingly, a dyad motif can be recognized in the structure of the C-terminus of MeuTXKβ5, in which a conserved Lys at site 22 and a hydrophobic residue Leu at site 31 can be well superimposed with that of ChTX (Fig. 5E).

Biochemical characterization and functional evaluation of MeuTXKα1 and BmP01
MeuTXKα1 and BmP01 are two orthologous toxins with accelerated amino acid substitutions, as identified by higher substitution rate in nonsynonymous sites of the mature peptide-coding region than in those of the signal peptide-coding region (Fig. S1, provided as Supplementary material). To study functional significance of the accelerated substitutions, we compared their channel-blocking activities. Firstly, we purified MeuTXKα1 from the M.   (Fig. 6A) and the MW detected is 3251 Da that accurately matches the predicted molecular weight (MW) from its amino acid sequence (3250 Da) (Fig. 6B). Edman degradation determined the N-terminal first five residues of the purified component which was VSCED, completely consistent with that of MeuTXKα1 determined by cDNA cloning. By using the same approaches, we also purified BmP01 from the M. martensii venom (Fig. 6C and D). All channels were expressed in Xenopus oocytes and their currents were recorded by using two-electrode voltage-clamp technique. Fig. 7 shows the blocking effects of MeuTXKα1 on different K + currents. At 3 µM concentration, MeuTXKα1 inhibited about 35%, 100%, and 70% of the peak currents of rKv1.1, hKv1.3 and Shaker IR channels, respectively. At this concentration, rKv1.2, rKv1.4, rKv1.5, rKv1.6, and hERG channels were not affected. For comparison, we also in parallel evaluated Kv channel-blocking activity of BmP01 on the same channels. The results showed that it exhibited an identical channel spectrum to MeuTXKα1 but was more potent on rKv1.1 than MeuTXKα1 because at 3 µM concentration, BmP01 inhibited 100% rKv1.1 currents (Fig. 8).

Discussion
M. eupeus is a sibling species of the most wide-studied species M. martensii (15), however, its KTxs are little-known. One such peptide previously isolated from this scorpion is BeKm-1, a human ether-a-go-go-related K + channel (hERG)-specific toxin, which shares structural similarity to ChTX but a similar mechanism of action to ErgTx, a member of the scorpion venom-derived γ-KTx family (27). Another KTx in this species (named MeuKTX) was recently identified as a non-selective inhibitor of Kv channels (28) Although as naturally-occurring bioactive components, scorpion venom-derived KTxs have shown highly potent activity in inhibiting Kv1.3, the majority of these peptides lack sufficient specificity to distinguish between this channel and other related Kv1.x, especially Kv1.1 given high-degree sequence similarity in the toxin-interacting pore region between Kv1.1 and Kv1.3 channels (30). For example, AgTx-2, OsK-1, NTX, and KTX bind to Kv1.3 with picomolar affinity, but their selectivity over Kv1.1 is very low, ranging from 2.6 to 110 fold (3, 31-33) ( Table 1). Other peptides, such as ADWX-1, HsTx1, Aam-KTX, Mokatoxin-1, and ChTX, possess high selectivity on Kv1.1, but they are also active on other related Kv channels (e.g. Kv1.2) (30,(33)(34)(35). In this aspect, MeuTXKα1 has more advantage than the peptides mentioned above in that it works at low nanomolar concentration but displays more than one thousand fold selectivity for Kv1.3 over Kv1.1. Importantly, it lacks activity on other related Kv channels even at micromolar concentrations.
Members in the α-KTx8 subfamily have been considered as relatively weak venom components due to their overall negatively charged surfaces. The discovery of high-affinity binding of MeuTXKα1 to Kv1.3 expands the pharmacological target of this unique subfamily.
In fact, all the members in the α-KTx8 subfamily have two identical residues (K 18 and N 21 , numbered according to MeuTXKα1) which are structurally equivalent to K 27 and N 30 in AgTx2 and many other Kv channel-targeted α-KTXs. In AgTx2, mutations of these two key residues had the largest destabilizing effects (36). Due to the conservation in these two key  (36). In our complex model, the region linking the α-helix and the first β-strand of the toxin approaches the turret, a known channel region responsible for high-affinity binding of ADWX-1 to Kv1.3 (38) and AgTx2 to Shaker (36), which could account for differential affinity between MeuTXKα1 and BmP01 because these two toxins have three amino acid substitutions in this region. To provide experimental evidence supporting the importance of the channel turret in interacting with the toxin, we compared the activity of BmP01 on hKv1.3 and rKv1.3, both differing by only two amino acids in the turret. The results showed that this toxin exhibited three-fold different affinity on human and rat Kv1.3 (Fig. 9), supporting the importance of the turret of Kv1.3 in toxin binding.
In conclusion, our work, which is based on cDNA cloning and biochemical purification and functional assays, describes molecular diversity of scorpion toxins affecting K + channels in a less studied species (M. eupeus) and functional diversification between orthologous             shown as mean ± SD (n = 3). Student's unpaired t-test for comparison of means was used to compare groups. ***P < 0.001 (compared with the control without peptides added). For clarity, only chains A and C of Kv1.3 are shown here; (D) BmP01 binds to the outer vestibule of Kv1.3 by K 18 plugging into the channel pore and two hydrophobic residues (A 1 and P 2 ) respectively interacting with V 406 derived from the chains A and C. In this mode, the turn between the α-helix and the first β-strand contains three non-identical residues between MeuTXKα1 and BmP01 and is adjacent to the channel turret. Amino acid color codes: blue: basic; green: hydrophobic; cyan: polar.    by guest on May 8, 2020