Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution

Snake venoms are complex mixtures of enzymes and non-enzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of non-enzymatic proteins. They share a common structure of three  -stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as  -bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs. SynTxs.


Purification and isolation of synergistic type toxin from Dendroaspis jamesoni jamesoni crude venom (Dj-SynTx).
The crude Dendroaspis jamesoni jamesoni venom 150 mg dissolved in 1 mL ultra-pure water (Milli-Q, Merck, Darmstadt, Germany), filtered and loaded to a HiLoad TM  Subsequently, iodoacetamide (final 15 mM) was added and the mixture was incubated at room temperature for 15 min. The alkylated protein was separated from the reaction mixture by RP-HPLC on a Jupiter C18 column (5 μm, 300 Å, 4.6 x 150 mm) using a linear gradient of 80% acetonitrile in 0.1% TFA. To determine of amino acid sequence using mass spectrometry, we generated short peptides that are easily amenable for sequencing. The alkylated protein was digested by each enzyme such as trypsin, endoproteinases Asp-N or Lys-C (sequencing grade) Fragmentation of peptides was accomplished by collision-induced dissociation using helium gas.
Product ions were scanned for 30 ms (3 μscans/25 ms), and dynamic exclusion was enabled in order to acquire sequences of all possible ions from the parent scan. The MS/MS data were analyzed using SEQUEST and Proteome Discoverer (Thermo Scientific) software. For more details, see [19].

Data collection and structure determination of the covalent dimer of Dj-SynTx.
The SynTx protein was concentrated to 8.0 mg/ml in phosphate-buffered saline ( [20]. There were two molecules of Dj-SynTx in the asymmetric unit. The Matthews coefficient was estimated to be 2.69 Å 3 /Da [21], corresponding to a solvent content of 54%. The structure was solved by Autorickshaw program [22] using Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200529/894232/bcj-2020-0529.pdf by guest on 06 October 2020 molecular replacement method. The coordinates of Muscarinic Toxin 2, which shows 61.5% identity and 75.4% similarity with Dj-SynTx (PDB Code: 1FF4) was used as the search model.
The model was built using the AutoBuild program [23], followed by manual model building using COOT program [24]. The structure was refined using Phenix-refine program [25]. The model has good stereochemistry, with 99.0% residues within the allowed regions of the Ramachandran plot analyzed by PROCHECK [26] (Table 1).

Results and Discussion
Purification of a synergistic-type toxin from African mamba snake.
We purified a synergistic-type toxin (Dj_SynTx) from D. jamesoni jamesoni venom by a two-step Three-dimensional structure of synergistic-type toxin.
The crystal structure of the native Dj_SynTx was determined to 2.4 Å resolution (  Figure S2). Both subunits have typical 3FTx structure with four conserved disulfide bonds and five -strands ( Figures 1C-D). The comparison of topologically similar protein structures with Dj_SynTx selected from Protein Data Bank (PDB) database by DALI program [27] showed the high similarity with neurotoxins and cardiotoxins belonging to 3FTx family for Dj_SynTx on almost less than 3.0 Å of RMSD although amino acid sequence identity are less than 50 % (Supplementary Table S1).
Three structural features contribute to the formation of unique quaternary structure of Dj_SynTx. The dimer is covalently held together by C54 A -C54 B interchain disulfide bond ( Figure   3A), and dimeric interfaces formed by direct interaction between the two subunits ( Figure 3B), and ionic and hydrogen bond interactions mediated through two sulfate ions ( Figure 3C).The antiparallel -sheet formed between loop III segments (Asp51-Lys56) brings the two monomers together through a series of hydrogen bonds, either directly between the -strands or mediated through water molecules and two hydrogen bonds between -N of Lys56 with O of -carbonyl of Asp51 (Table 2), leading to the covalent C54 A -C54 B disulfide bond ( Figure 3A). The dimeric interface is formed by hydrogen bonds and electrostatic interactions between Trp28 and Lys34 of one subunit with Tyr36 and Asp37 of the other subunit, respectively ( Figure 3A, Table 2).
There are two --interactions (3.8 Å) between Trp28 from chain A and Tyr36 from chain B residues and vice versa. In addition, the dimer is held together by two sulfate ion-mediated interactions ( Figure 3C, Table 3). Each sulfate ion is electrostatically caged by Lys30, Lys34, Leu35 and Tyr36 from one subunit and Lys7 and Arg40 from the other subunit. Sulfate ions exist in human blood (~0.5 mM), which are essential for the synthesis of many biomolecules such as glycosaminoglycans, choline sulfate, steroid sulfate, cerebroside sulfate and heparan sulfate [28]. Although crude venom contains inorganic salts, there is documented evidence for the presence of sulfate. It is unclear whether the sulfate ions are from Dendroaspis venoms or are incorporated in the structure during crystallization. Of the 1164 Å 2 of the buried surface area Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200529/894232/bcj-2020-0529.pdf by guest on 06 October 2020 during the dimer formation, 25% is formed by hydrophobic residues, 39% by polar residues, and 36% by charged residues. Most amino acid residues involved in dimerization are found on the concave surface of the monomers (Supplementary Figures S3 and S4). All these residues are conserved in mamba SynTx family of 3FTxs ( Figure 1B).

Quaternary structures of dimeric 3FTxs.
The -neurotoxins, a family of 3FTxs all isolated from Bungarus (krait) venoms, are the first group of homodimers of 3FTxs [7] (Figure 4). They bind to a variety of neuronal nAChRs [8] but bind reversibly and with weak affinity to muscle-type peripheral nAChR [29]. -bungarotoxins show similarity to long-chain -neurotoxins that have the fifth disulfide in loop II but without the C-terminal extension. In the dimer, the three-stranded -sheet of the monomer is extended to a six stranded -sheet and the two monomers are antiparallel to each other [30]. Haditoxin from Ophiophagus hannah (king cobra) venom is a homodimer of short-chain type 3FTxs that does not have the fifth disulfide bond [9]. It interacts with neuronal 7 and muscle-type nAChRs.
Haditoxin shares similar quaternary structure as the -neurotoxins. The antiparallel dimeric interface is maintained by six main chain-main chain hydrogen bonds (9,20). -bungarotoxin and haditoxin have three and eight side chain hydrogen-bonding contacts between the monomers. Fulditoxin, the first member of -neurotoxins from Micrurus (coral snake) venoms, is also a noncovalent homodimer of short-chain type 3FTxs [10]. It binds to chicken muscle-type receptor with high potency compared to cloned human receptor. Fulditoxin has a distinct quaternary structure; the two subunits intertwine each other through hydrophobic and hydrogen bond interactions. Thus, at least two distinct types of noncovalent dimers have evolved ( Figure   4).
All three covalent dimers have distinct quaternary structures ( Figure 4) and hence, they probably evolved independently. In -cobratoxin homodimers from Naja kaouthia venom, the Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200529/894232/bcj-2020-0529.pdf by guest on 06 October 2020 subunits are covalently linked through two disulfide bonds; the loop I segments of both subunits stretch to form new antiparallel -sheet with that of the other subunit ( Figure 5) [12]. Normally, this long-chain -neurotoxin occurs as a monomer ( Figure 5-'a'). With the change from Cis to Trans isomerization of X-Pro7 peptide bond, the loop I opens and extends the N-terminal segment away [12] from the core of the protein ( Figure 5-'b'). There is significant entropic cost for unfolding of loop I. The newly exposed surface is 'buried' through the second monomer with similar extended conformation forming two interchain disulfide bonds (C1 A -C3 B and C3 A -C1 B ) ( Figure 5-'c') and form this dimer. The high entropic cost of the extension of the N-terminal is probably the reason why this dimer is only 0.04% of the venom [9] compared to 15-20% of the monomer [31]. In the case of irditoxin isolated from Boiga irregularis (Brown tree snake) venom, the heterodimer is covalently held together by a single disulfide bond [13]. Both subunits show similarity to nonconventional toxins with the fifth disulfide bond in the loop I. Unlike other nonconventional toxins, the subunits of this colubrid toxin have the extraordinarily 7-residue long, unstructured NH 2 terminal segments that are located above the core. The interchain disulfide bond linkage is between Cys in loop II of A subunit with Cys in loop I of B subunit and the subunits are placed in a diagonal geometry [13]. As described above, Dj_SynTx dimer has elaborate interactions in dimer formation. Interestingly, the disulfide linkage is driven by similar antiparallel -sheet formation that is involved in -neurotoxins and haditoxin. We speculate that the interchain disulfide linkage followed by the evolution of other interactions between the subunits results in the unique quaternary structure of SynTxs (see below).

Molecular evolution to unusual quaternary structure of SynTx dimers.
A careful evaluation of dimeric interfaces indicates that SynTx quaternary structure evolved systematically from noncovalent dimers ( Figure 6). Although most 3FTxs are monomers in solution ( Figure 6-'a'), some 3FTxs crystallize as noncovalent dimers [34] (Figure 6-'b'). Such dimers have similar antiparallel -sheet similar to -bungarotoxins. Two pairs of electrostatic interactions (Glu54 A -Lys56 B and Lys56 A -Glu54 B in -elapitoxin-Dpp2d) also contribute this 'transient' dimer formation ( Figure 6A). With slightly increased salt concentration (isosmotic), the dimer breaks down to monomers [34]. Thus, this group of 3FTxs are monomeric at physiological conditions. This dimeric interface is stabilized further with additional noncovalent (hydrophobic and electrostatic) interactions in -bungarotoxins and haditoxin [9,30] (Figures 6-'c', 7B). These 3FTxs remain as dimers even at high salt concentrations. However, when -bungarotoxin is mixed with-flavitoxin, due to dissociation and association kinetics, they form a mixture of homo-and bungarotoxin-flavitoxin hetero-dimers [35]. Thus, there is an equilibrium state in which the molecules remain in the dimeric forms but not in monomeric forms. In SynTxs, all the residues involved in the dimerization are fully conserved ( Figure 1B). The cysteine in loop III segment is involved in interchain disulfide bond that covalently links the monomers (Figure 6-'d').
This contortion leads to a slight shift of the antiparallel -sheet by two amino acid residues towards the N-terminal side ( Figure 7C). Thus, we propose that the unique dimers of SynTxs have evolved from monomeric 3FTXs through noncovalent dimers like haditoxin and bungarotoxins ( Figure 6). To our knowledge, this is the first instance in which the evolution of a quaternary structure is determined through the three-dimensional structure.
The other 3FTx dimers all exhibit postsynaptic neurotoxicity by binding to acetylcholinebinding pocket of nicotinic acetylcholine receptors. Dimerization changes their subtype selectivity (discussed above). Unlike other dimers of 3FTxs, SynTxs have acquired ability to synergistically enhance the toxicity of other snake venom toxins. Interestingly, the electrostatic charge distribution in Dj_SynTx (Supplementary Figure S5) indicates that the positive and negative charges cluster on both surfaces of the molecule. This structure will help in understanding the structure-function relationships and mechanism of action of this unique class of 3FTxs including mechanism of synergistic function.

Data Availability
Coordinates and structure factors of the Synergistic-type toxin are deposited in PDB (Protein Data Bank) with accession code 7C28.

Competing Interests
Authors declare that there are no competing interests with the data described in this manuscript.     In the -cobrotoxin monomer 'a', Pro7 has Cis peptide bond resulting turn leading to loop I formation. When the this Pro7 has Trans peptide bond, the loop I opens and extends the Nterminal segment away [12] from the core of the protein 'b'. This will double the exposed surface area. The newly exposed surface is 'buried' through the second monomer with similar extended conformation. Two interchain disulfide bonds (C1 A -C3 B and C3 A -C1 B ) are formed 'c', instead of intrachain disulfide bond (C1-C3) within each monomer. We propose that higher entropic cost in such extended N-terminal limits its occurrence to very low levels (0.04% of the venom) [9]. Most 3FTxs exist as monomers 'a', Some of monomers evolved to form transient 'b' or stable 'c' noncovalent dimers through the formation of antiparallel -sheet between loop III segments of two monomers. They are held together by hydrogen bonds between the -strands and a small number of electrostatic and/or hydrophobic interactions ( Figure 6). Even in the stable noncovalent dimers, the subunits can interchange to form homo-or hetero-dimers [27]. Some of these dimers have evolved to have a disulfide bond to covalently hold the dimeric structure 'd'.
With further dimeric interactions, the monomers twist and contort across the disulfide bond to form a stable quaternary structure of SynTxs 'e'. (A) Some 3FTxs, such as-elapitoxin-Dpp2d, form dimers through antiparallel -sheet between two monomers during crystallization [26]. In addition to hydrogen bonds, sometimes electrostatic interactions (highlighted in blue) also contribute the noncovalent interaction. At isosmotic conditions, the transient dimer breaks into stable monomers [26]. (B) Stable dimers are fortified by additional interactions between these-strand and elsewhere (for details, see [9,22]). Because of the noncovalent nature, the subunits interchange forming homo-or heterodimers [27]. (C) A newly formed interchain disulfide in this segment covalently holds the monomers together. The Cys residue involved in this disulfide is identified by a red triangle.
Because of the contortions (Figure 5), the antiparallel-sheet is 'shifted' two residues towards the N-terminal (indicated by blue arrow). Thus, monomeric 3FTxs have evolved into noncovalent dimers and subsequently into covalent dimeric organization of SynTx.