RESUMEN
alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.
Asunto(s)
Proteínas Neurotóxicas de Elápidos/química , Proteínas Neurotóxicas de Elápidos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Sitios de Unión , Dicroismo Circular , Proteínas Neurotóxicas de Elápidos/genética , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Fragmentos de Péptidos/química , Unión Proteica , Conformación Proteica , Receptores Nicotínicos/química , Proteínas Recombinantes de Fusión/metabolismo , TorpedoRESUMEN
Snakes and cone snails produce toxins which block muscular and/or neuronal nicotinic acetylcholine receptors (AChRs). This paper mostly focuses on the determinants by which a snake long chain curaremimetic toxin and the cone snail toxin ImI bind specifically to the alpha 7 neuronal receptor. In both cases, the site involves a small turn-like structure constrained by two half-cystines.
Asunto(s)
Conotoxinas , Venenos de Moluscos/química , Neuronas/fisiología , Antagonistas Nicotínicos/química , Oligopéptidos/química , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Venenos de Serpiente/química , Secuencia de Aminoácidos , Animales , Caribdotoxina/química , Caribdotoxina/farmacología , Curare/análogos & derivados , Curare/química , Curare/farmacología , Erabutoxinas/química , Erabutoxinas/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Venenos de Moluscos/síntesis química , Venenos de Moluscos/farmacología , Mutagénesis Sitio-Dirigida , Antagonistas Nicotínicos/síntesis química , Antagonistas Nicotínicos/farmacología , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Caracoles , Venenos de Serpiente/síntesis química , Venenos de Serpiente/farmacología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60-62 residues and four disulfide bonds) and long-chain toxins (66-74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine alpha7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.