The following are available online at
https://www.mdpi.com/2072-6651/10/5/172/s1, Figure S1: Three disulfide bond patterns are evident among 8-cysteine 3FTxs from the venoms of
Micrurus corallinus,
M. l. carvalhoi,
M. l. lemniscatus,
M. paraensis,
M. spixii, and
M. surinamensis. The first pattern is illustrated by sequences 1–12, and includes the overwhelming majority of 8-Cys toxins. The second, represented by sequences 13–19, occurs in
M. l. carvalhoi,
M. paraensis,
M. lemniscatus, and
M. surinamensis. These toxins have four cysteines among the N-terminal 18 amino acids, like γ-bungarotoxin (positions 3, 6, 11, and 17), and they lack the two C-terminal cysteines (positions 65 and 70), including one of the usual paired residues. The third pattern is represented by sequences 57–60 and 92. In these toxins, the paired cysteines have become separated, with Cys-66 being displaced two residues C-terminally, such that the three C-terminal cysteines occur in positions 64, 67, and 70, instead of 64, 65, and 70. The functional significance of these patterns is unknown. A tree was constructed using the Neighbor-Joining method with the Jukes-Cantor model and γ-bungarotoxin from
Bungarus multicinctus venom as an outgroup. Asterisks indicate stop codons, which were paired in some cases; Figure S2: 3FTxs having 9 cysteines, derived from venoms of
Micrurus corallinus,
M. l. carvalhoi,
M. l. lemniscatus,
M. paraensis,
M. spixii, and
M. surinamensis, display two disparate disulfide bond patterns. The first, representing the majority of 9-Cys toxins, has paired second and third cysteines in positions 16–17 (sequences 1–28 and 30–31). In the second group, the cysteine in position 16 is absent and instead a cysteine is present in position 63. Whether the extra cysteine is free in monomeric toxins, or whether these 9-Cys toxins form homodimers, or heterodimers with a non-homologous toxin is unknown. γ-bungarotoxin from
B. multicinctus venom was used as an outgroup. Asterisks indicate stop codons; Figure S3: Venoms of
Micrurus corallinus,
M. l. carvalhoi,
M. l. lemniscatus,
M. paraensis,
M. spixii, and
M. surinamensis all have 3FTxs with 10 cysteines and our samples of
M. l. carvalhoi and
M. spixii also have novel toxins with an eleventh cysteine occurring at either position 32 or 37. The functional significance of these patterns is unknown. A tree was constructed using the Neighbor-Joining method with the Jukes-Cantor model and γ-bungarotoxin from
B. multicinctus venom as an outgroup. Asterisks indicate stop codons; Figure S4: South American
Micrurus 3FTx sequences comprise as astonishing variety of structural subtypes, including many that have not been reported from the North American species,
M. fulvius and
M. tener. However, some subtypes are common to both groups. Previously published 3FTx sequences were downloaded from the NCBI nr database. γ-bungarotoxin from
B. multicinctus venom was used as an outgroup. Asterisks indicate stop codons; Figure S5: The
Micrurus taxa investigated here rely much less heavily on PLA
2 toxins than do the North American taxa,
M. fulvius and
M. tener. Still, various structural subclasses are evident, although differences in function cannot be surmised at present, except for probable non-catalytic PLA
2s that lack the active site His-58 and/or Asp-59 residues (see main text). Most of the North American sequences cluster separately from the Brazilian sequences. PLA
2 sequences from Brazilian
Micrurus species were identified with Megablast, using nine complete PLA
2s, including signal peptides, as queries. 35 Brazilian
Micrurus PLA
2s are aligned here with 169 published sequences, using Geneious software. A tree was constructed using the Neighbor-Joining method with the Jukes-Cantor model, with
Bungarus fasciatus PLA
2 BF-32 as an outgroup. Signal peptides are not shown in this alignment. Asterisks indicate stop codons.
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