Variations in "Functional Site" Residues and Classification of Three-Finger Neurotoxins in Snake Venoms

Abstract

Three-finger toxins (3FTxs) are the largest group of nonenzymatic toxins found in snake venoms. Among them, neurotoxins that target nicotinic acetylcholine receptors are the most well-studied ligands. In addition to the classical neurotoxins, several other new classes have been characterized for their structure, receptor subtype, and species selectivity. Here, we systematically analyzed over 700 amino acid sequences of three-finger neurotoxins that interact with nicotinic acetylcholine receptors. Based on the amino acid residue substitutions in the functional sites and structural features of various classes of neurotoxins, we have classified them into over 150 distinct subgroups. Currently, only a small number of typical examples representing these subgroups have been studied for their structure, function, and subtype selectivity. The functional site residues responsible for their interaction with specific receptor subtypes of several toxins are yet to be identified. The molecular details of each subgroup representative toxin with its target receptor will contribute towards the understanding of subtype- and/or interface-selectivity. Thus, this review will provide new impetus in the toxin research and pave the way for the design of potent, selective ligands for nicotinic acetylcholine receptors.

Keywords: ligand-gated ion channels; molecular evolution; nicotinic acetylcholine receptor (nAChR); phylogenic analysis; protein–protein interaction; sequence alignment; structure–function relationships.

Figure 1

(a) Sequence alignment of Type I (short-chain) Class 1 neurotoxins. This class of neurotoxins is divided into nine groups. (b) Sequence alignment of Type I (short-chain) Class 2 to 9 neurotoxins. Functional residues, colored red, are based on mutagenesis studies on the binding of erabutoxin to the peripheral nAChR of the Torpedo electric ray. Erabutoxin, with Ile³⁶⁽⁴⁰⁾ (green), gained binding to the receptor when mutated to Arg³⁶⁽⁴⁰⁾ (black, bold). Sequences in this class of neurotoxins have a bulky, hydrophobic residue (green) or a positively charged residue (black, bold) at this position. Conserved disulfide-forming Cys are highlighted yellow. The four disulfide bonds are indicated above the sequences in red, black, green, and blue. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group.

Figure 1

(a) Sequence alignment of Type I (short-chain) Class 1 neurotoxins. This class of neurotoxins is divided into nine groups. (b) Sequence alignment of Type I (short-chain) Class 2 to 9 neurotoxins. Functional residues, colored red, are based on mutagenesis studies on the binding of erabutoxin to the peripheral nAChR of the Torpedo electric ray. Erabutoxin, with Ile³⁶⁽⁴⁰⁾ (green), gained binding to the receptor when mutated to Arg³⁶⁽⁴⁰⁾ (black, bold). Sequences in this class of neurotoxins have a bulky, hydrophobic residue (green) or a positively charged residue (black, bold) at this position. Conserved disulfide-forming Cys are highlighted yellow. The four disulfide bonds are indicated above the sequences in red, black, green, and blue. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group.

Figure 2

(a) Sequence alignment of Type II (long-chain) Class 1 and Class 2, Group 2a neurotoxins. (b) Sequence alignment of Type II (long-chain) Class 2, Groups 2b and 2c neurotoxins. (c) Sequence alignment of Type II (long-chain) Classes 3 to 6 neurotoxins. Functional residues, colored red, are based on mutagenesis studies on the binding of α-cobratoxin to the peripheral nAChR of the Torpedo electric ray. Conserved disulfide-forming Cys are highlighted yellow. The five disulfide bonds are indicated above the sequences in red, black, green, blue, and orange. In Class 6 sequences, intermolecular disulfides were indicated by purple line, with dashed line indicating inferred disulfides. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group.

Figure 2

(a) Sequence alignment of Type II (long-chain) Class 1 and Class 2, Group 2a neurotoxins. (b) Sequence alignment of Type II (long-chain) Class 2, Groups 2b and 2c neurotoxins. (c) Sequence alignment of Type II (long-chain) Classes 3 to 6 neurotoxins. Functional residues, colored red, are based on mutagenesis studies on the binding of α-cobratoxin to the peripheral nAChR of the Torpedo electric ray. Conserved disulfide-forming Cys are highlighted yellow. The five disulfide bonds are indicated above the sequences in red, black, green, blue, and orange. In Class 6 sequences, intermolecular disulfides were indicated by purple line, with dashed line indicating inferred disulfides. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group.

Figure 2

(a) Sequence alignment of Type II (long-chain) Class 1 and Class 2, Group 2a neurotoxins. (b) Sequence alignment of Type II (long-chain) Class 2, Groups 2b and 2c neurotoxins. (c) Sequence alignment of Type II (long-chain) Classes 3 to 6 neurotoxins. Functional residues, colored red, are based on mutagenesis studies on the binding of α-cobratoxin to the peripheral nAChR of the Torpedo electric ray. Conserved disulfide-forming Cys are highlighted yellow. The five disulfide bonds are indicated above the sequences in red, black, green, blue, and orange. In Class 6 sequences, intermolecular disulfides were indicated by purple line, with dashed line indicating inferred disulfides. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group.

Figure 3

Sequence alignment of Type III neurotoxins. Functional residues are not known. Potential amidation for Gly at C-terminal in the lone sequence in Class 2 is highlighted in magenta. Conserved disulfide-forming Cys are highlighted yellow. The four disulfide bonds are indicated above the sequences in red, black, green, and blue. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group.

Figure 4

Sequence alignment of non-conventional neurotoxins. Compared to erabutoxin, functional residues that are conserved are colored red. Based on mutagenesis of WTX (Class 3) binding to nAchRs, Arg³¹⁽³³⁾ and Arg³²⁽³⁷⁾ residues, colored green, are important functional residues. A positively charged residue at position 42 (bold, black) is potentially a specific determinant for interaction with mAChRs but not nAChRs. The integrin-binding tripeptide RGD in Class 2 sequences is colored blue. Conserved disulfide-forming Cys are highlighted yellow. The four disulfide bonds are indicated above the sequences in red, black, green, and blue.

Figure 5

Sequence alignment of Ω-neurotoxins. Functional residues, colored red, are based on mutagenesis studies for the binding of Oh9-1 to rat α1β1εδ. Mutation of Thr²⁴⁽²⁹⁾ and Tyr⁴⁶⁽⁵⁴⁾, colored green, leads to increased inhibition against rat α3β2 nAChR. Conserved disulfide-forming Cys are highlighted yellow. The four disulfide bonds are indicated above the sequences in red, black, green, and blue.

Figure 6

Sequence alignment of Σ-neurotoxins. Functional residues are not known. Fulditoxin is not amidated at C-terminal despite the presence of Gly (highlighted in magenta). Residues involved in dimerization are highlighted in green. Zn²⁺-binding His that facilitates tetramerization of dimers is highlighted in cyan. Conserved disulfide-forming Cys are highlighted yellow. The four disulfide bonds are indicated above the sequences in red, black, green, and blue.

Figure 7

(a) Sequence alignment of colubrid neurotoxins, Class 1. (b) Sequence alignment of colubrid neurotoxins Classes 2 to 7. (c) Sequence alignment of colubrid neurotoxins Classes 8 to 11. Functional residues are not known. Propeptide region is shaded in gray, with potential basic processing sites highlighted in cyan. N-terminal Gln in Class 1 colubrid neurotoxins, colored red, are post-translationally modified to pyroglutamic acid. Conserved loop II residues CYTLY and WAVK are identified to play a key role in avian/reptilian-selective neurotoxicity (underlined). Non-Ala residues within the WAVK segment are highlighted in black. In Denmotoxin, Pro⁴⁰⁽⁵⁵⁾ (colored blue) caused a twisted tip of the central loop. The crucial positively charged loop II tip residue (typically Arg) in short- and long-chain α-neurotoxins is replaced by Asp⁴¹⁽⁵⁶⁾ (colored red). This residue may bind to Arg193 of the δ-subunit to determine species selectivity. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group. Conserved disulfide-forming Cys are highlighted yellow. The five disulfide bonds are indicated above the sequences in red, black, green, blue, and orange. Cys residues forming intermolecular disulfide (magenta line) in Classes 8 and 9 are colored red with yellow highlight.

Figure 7

(a) Sequence alignment of colubrid neurotoxins, Class 1. (b) Sequence alignment of colubrid neurotoxins Classes 2 to 7. (c) Sequence alignment of colubrid neurotoxins Classes 8 to 11. Functional residues are not known. Propeptide region is shaded in gray, with potential basic processing sites highlighted in cyan. N-terminal Gln in Class 1 colubrid neurotoxins, colored red, are post-translationally modified to pyroglutamic acid. Conserved loop II residues CYTLY and WAVK are identified to play a key role in avian/reptilian-selective neurotoxicity (underlined). Non-Ala residues within the WAVK segment are highlighted in black. In Denmotoxin, Pro⁴⁰⁽⁵⁵⁾ (colored blue) caused a twisted tip of the central loop. The crucial positively charged loop II tip residue (typically Arg) in short- and long-chain α-neurotoxins is replaced by Asp⁴¹⁽⁵⁶⁾ (colored red). This residue may bind to Arg193 of the δ-subunit to determine species selectivity. The green highlight denotes an amino acid substitution with properties distinct from typical sequences in the group. Conserved disulfide-forming Cys are highlighted yellow. The five disulfide bonds are indicated above the sequences in red, black, green, blue, and orange. Cys residues forming intermolecular disulfide (magenta line) in Classes 8 and 9 are colored red with yellow highlight.

Figure 8

Gene organization of colubrid neurotoxins. (a) Compared to elapid and viperid 3FTxs, Class 1 colubrid neurotoxins, represented by denmotoxin, have an exon insertion (exon 2, pink) resulting in a propeptide segment in translated protein. (b) Exon 2 insertion can be observed in most classes of colubrid neurotoxins, resulting in at least 10 distinct propeptide segments. N-terminal Gln that may be modified to pyroglutamic acid is colored red. Propeptide region is shaded gray. Potential basic propeptide processing site are highlighted with cyan. Conserved disulfide-forming Cys are highlighted yellow.

Figure 9

Structural alignment of Type I (pink), Type II (cyan), and non-conventional (green) neurotoxins showing functional residues, many of which are topologically conserved. Residues are numbered by homology numbering indicated in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7. PDB id for erabutoxin: 1QKD; α-cobratoxin: 7ULG; candoxin: 1JGK.

Figure 10

Structural alignment of Type I neurotoxins. (a) Superpositions of selected Class 1 Group 1 neurotoxins showing structural variations in loop III introduced by flanking Pro50 and Pro54. Changes in the number of residues and the presence of Pro also introduce variations in the connecting loop between loops I and II, although the functional effect of such changes may be minimal since this site is not directly involved in binding to nAChRs. Variations in the tip of loop II potentially contribute to nAChR subtype selectivity. Class 1 Group 1a is pink, Class 1 Group 1b (iii) is cyan, Glass 1 Group 1b (iv) is green, Class 1 Group 1c (ii) is purple, Class 1 Group 1d (i) is blue, Class 1 Group 1e is brown, Class 1 Group 1f is orange. (b) Superpositions of selected Class 1 Group 2 neurotoxins showing the effect of length and Pro in the connecting loop between loops I and II. Notably, Class 1 Group 2e has a longer loop II. Class 1 Group 2b is pink, Class 1 Group 2c is cyan, Class 1 Group 2e is green. (c) Superpositions of selected Class 1 Group 6 neurotoxins showed that the presence of flanking Pro in loop I affects the conformation of the loop. Class 6 Group 6a is pink, Class 6 Group 6b is cyan, Class 6 Group 6c is green, Class 6 Group 6d is purple. (d) Superpositions of selected Class 5 to 8 neurotoxins demonstrating diversity in the length of loops I and II. The crystal structure of a Class 6 Group 6a neurotoxin showed that the longer loop II has a similar conformation to that of a prototypical Type II neurotoxin (e.g., α-bungarotoxin/α-cobratoxin). Class 5 Group 5a is orange, Class 6 Group 6a is pink, Class 7 Group 7 is cyan, Class 8 Group 8 is green. Residues are numbered by homology numbering indicated in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7. PDB id for erabutoxin: 1QKD; Class 6 Group 6a: 8D9Y; structures of all other neurotoxins were predicted by AlphaFold3.

Figure 11

Structural alignment of Type II neurotoxins. (a) Superpositions of selected Class 1 Group 1 neurotoxins showing most variations occurred in loop III flanked by Pro50 and Pro54. Class 1 Group 1a (i) is pink, Class 1 Group 1a (ii) is cyan, Class 1 Group 1a (iii) is green, Class 1 Group 1a (iv) is purple. (b) Superpositions of selected Class 1 Group 1 neurotoxins showing a single residue deletion in loop I may drastically affect the conformation of the loop. Class 1 Group 1b is blue, Class 1 Group 1c is brown. (c) Superpositions of selected Class 1 Group 3 neurotoxins demonstrating the effect of variation in loop I size and flanking Pro in loop III. These neurotoxins also have long and variable C-termini. Class 1 Group 3a is pink, Class 1 Group 3b is cyan, Class 1 Group 3c (i) is green. (d) Superpositions of selected Class 1 Group 2 neurotoxins similarly showing the effect of variation in loop I size and flanking Pro. Variations in the tip of loop II potentially contribute to nAChR subtype selectivity. Class 1 Group 2a (i) is pink, Class 1 Group 2a (iia) is cyan, Class 1 Group 2a (iiib) is green, Class 1 Group 2a (iv) is grey, Class 1 Group 2a (va) is purple. (e) In some sequences, Pro in loop I further influences structural variation. Class 1 Group 2b (i) is blue, Class 1 Group 2b (ii) is brown, Class 1 Group 2b (iii) is lime. (f) Changes in the number of residues and the presence of Pro also introduce variations in the connecting loop between loops I and II, although the functional effect of such changes may be minimal since this site is not directly involved in binding to nAChRs. Class 1 Group 2c (iia) is pale blue, Class 1 Group 2c (ix) is wheat, Class 1 Group 2c (xi) is light green. Residues are numbered by homology numbering indicated in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7. PDB id for α-bungarotoxin: 1HC9; structures of all other neurotoxins were predicted by AlphaFold3.

Figure 12

Conservation of functional residues in Type I and Type II neurotoxins. (a) Crystal structure of Type 1 Class 6 Group 6a (pink) superposes well on Type II (long chain, grey) neurotoxin, despite the missing ‘canonical’ fifth disulfide bridge in loop II. Many functional site residues in both these structures are topologically well conserved. (b) Most functional site residues in Type 1 Class 5 Group 5a (orange) neurotoxin superpose well on α-cobratoxin (cyan) residues, despite the absence of half-helix structure imposed by the fifth disulfide. Residues are numbered by homology numbering indicated in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7. PDB id for α-bungarotoxin: 1HC9; erabutoxin: 1QKD; α-cobratoxin: 7ULG; Type 1 Class 6 Group 6a: 8D9Y; structure of Type 1 Class 5 Group 5a was predicted by AlphaFold3.