Synthesis, H-1 NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the veno m of the chactidae scorpion Scorpio maurus palmatus, The toxin displays an exceptionally wide range of pharmacological activity since it binds onto sm all conductance Ca2+-activated K+ channels and also blocks Ky channels (Sha ker, Kv1.2 and Kv1.3). MIX possesses 53-68% sequence identity with HsTx1 an d Pi1, two other Kf channel short chain scorpion toxins cross-linked by fou r disulfide bridges. These three toxins differ from other K+/Cl-/Na+ channe l scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide b ridges). Because MTX exhibits an uncommon disulfide bridge organization amo ng known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically s ynthesized an MTX analog with three instead of four disulfide bridges ([Abu (19),Abu(34)]MTX and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (A bu) derivatives. The three-dimensional structure of [Abu(19),Abu(34)]MTX in solution was solved by H-1 NMR, This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C 3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement i n half-cystine pairings that concerns the last disulfide bridge results mai nly in a reorientation of the alpha-helix regarding the beta-sheet structur e. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intr acerebroventricular injection of the peptide (LD50 value of 0.25 mu g/mouse ). The structural variations are also accompanied by changes in the pharmac ological selectivity of the peptide, suggesting that the organization patte rn of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K+ channel subtypes.