On the Convergent Evolution of Animal Toxins

Abstract

BgK is a K؉ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ؎ 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K ؉ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the threedimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.Functional properties of proteins are frequently associated with a small number of important residues. For example, enzyme activities depend on a few residues that are essential for catalysis. Also, protein-protein recognition processes have been predicted (1) and recently demonstrated (2) to be energetically driven by a small proportion of the residues forming the contacting areas in protein-protein complexes, as identified by x-ray studies (3, 4). Among the proteins whose major functions require protein-protein interactions are animal toxins, which bind to various molecular targets, such as receptors or ion channels, using a small number of binding residues (5-8). As has been shown for enzymes (9), toxins with different architectures are capable of exerting similar functions (10). However, in contrast to enzymes, the molecular basis associated with the conservation of the function in structurally unrelated toxins remains unknown. In this paper, we show that two families of animal toxins with different folding patterns but a comparable capacity to bind to potassium channels include similar functional diads, composed of a critical lysine and an aromatic amino acid separated from each other by 6.6 Ϯ 1.0 Å. MATERIALS AND METHODS Synthesis of Toxin and Mutants-The amino acid sequence of BgK 1 was proposed a few years ago (11). However, chemical synthesis attempts, based on these data, systematically failed. The proposed amino acid sequence was therefore questioned, re-examined, and ultimately corrected.2 The revised amino acid sequence of BgK from Bunodosoma granulifera is: VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTC-ELC. BgK and each alanine-substituted analog were synthesized by solid phase synthesis using an Applied Biosystems model 431A peptide synthesizer, starting from 0.1 mmol of Rink-resin (4-(2Ј,4Ј-dimethoxyphenylhydroxymethylphenoxy resin; 0.48 mmol/g). A 10-fold excess (1 mmol) of Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acid was used and coupled in N-methylpyrrolidone in the presence of N,NЈ-dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The following side chain protections wer...

A Potassium‐Channel Toxin From the Sea Anemone Bunodosoma Granulifera, An Inhibitor for Kv1 Channels — Revision of the Amino Acid Sequence, Disulfide‐Bridge Assignment, Chemical Synthesis, and Biological Activity

“…Also ShK and kaliseptine seem to possess the same secondary structure as BgK, since their far-ultraviolet CD spectra (not shown) are superimposable with that of BgK. Recently, the solution structure of ShK has been reported [58] and indeed, the fold adopted by this toxin closely resembles that of BgK, whose three-dimensional structure has been elucidated by Dauplais et al [59], using 'H-NMR and modelling. Sequence similarity, identical disulfide pattern, similar secondary structure and identical folds show that sea anemone K' channel toxins represent a new family of structurally related peptide toxins, exhibiting diverse specificity for K' channels, as with K' channel inhibitors from scorpion venoms.…”

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

“…All sea anemone toxins contain a lysine residue (Lys 25 in BgK, Lys 22 in ShK) that occludes the K ϩ channel pore (16,(51)(52)(53)(54). Replacement of this residue with alanine abolishes K ϩ channel blocking activity, whereas replacement with shorter or longer chained positively charged residues retains activity, albeit with lower potency (51)(52)(53)(54).…”

“…Indeed, venomous creatures possess toxins with homology to several proteins, including acetylcholinesterases (5), phospholipases (6, 7), nerve growth factor (8), endothelins (9), Lynx-1 (10, 11), Kunitz-type serine protease inhibitors (12), and the ion channel regulatory (ICR) 5 domains of cysteinerich secretory proteins (CRISPs) (3,13,14). Mammalian proteins containing toxin-like domains (TxDs) that block K ϩ channels have not been characterized previously.BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera (15,16), and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus (17,18) are potent inhibitors of K ϩ channels. The Simple Modular Architecture Research Tool (SMART) (available on the World Wide Web) predicts the existence of a large superfamily of proteins that contain domains (referred to as ShKT domains in the SMART data base) resembling these two toxins (Fig.…”

“…BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera (15,16), and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus (17,18) are potent inhibitors of K ϩ channels. The Simple Modular Architecture Research Tool (SMART) (available on the World Wide Web) predicts the existence of a large superfamily of proteins that contain domains (referred to as ShKT domains in the SMART data base) resembling these two toxins (Fig.…”

Design and Characterization of a Highly Selective Peptide Inhibitor of the Small Conductance Calcium-activated K+Channel, SkCa2

“…Peptide inhibitors of SK Ca channels form a structurally distinct group and lack the conserved dyad present in peptide blockers of K V , IKCa1, and BK Ca channels that consists of an aromatic residue with a neighboring invariant lysine (43). Therefore, the architecture of the toxin-binding surface in the external vestibule of SK Ca channels may differ from that of K V (44 -47) and IKCa1 (28, 48) channels, which are similar to that of the crystallographically defined structure of the bacterial K ϩ channel, KcsA (49,50).…”