Disulfide bridge reorganization induced by proline mutations in maurotoxin

Abstract

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfidebridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1^C5, C2Ĉ6, C3^C4 and C7^C8, versus C1^C5, C2^C6, C3^C7 and C4Ĉ 8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys 13 ) and C4 (Cys 19 ), results in conventional Pi1-and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A 12 ,A 20 ]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog. ß

A Maurotoxin with Constrained Standard Disulfide Bridging

“…However, MTX is able to adopt the standard C1-C5, C2-C6, C3-C7, and C4-C8 arrangement (as observed in other ␣-KTx6 members) consecutive to selective amino acid residue substitution(s) (16 -18). The latter concerned Lys 15 , Gly 33 (18), or Pro 12 and Pro 20 (17) of MTX and were found to markedly affect peptide affinities toward the various K ϩ channel subtypes. Though the substituted residues were mainly selected on the basis of a pre-* This work was supported by CNRS, INSERM, the Commissariat à l'Energie Atomique, and Cellpep S.A.…”

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

“…MTX Pi1 blocks, effectively Shaker B K + , outward currents with higher affinity than MTX7. Besides obtaining this analog through Tyr 32 modification, we also observed that similar shifts in half‐cystine pairings could be produced by introducing selected point mutations in MTX amino acid sequence8–10. The MTX Pi1 pattern is the native pattern of the MTX‐related toxin, such as Pi111 and HsTx112, both short‐chain four disulfide‐bridged scorpion toxins sharing a high degree of sequence identity with MTX (from 53% to 68%).…”

“…The two Pro residues were chosen because of the structural constraints they induce in the MTX structure, suggesting that they were susceptible to influence MTX disulfide bridge pattern. As a matter of fact, the double mutation Ala 12 and Ala 20 was shown to produce the Pi1‐like disulfide bridging10. Gly 33 was chosen because of its strategic location between Cys 31 and Cy 34 , also suggesting its potential involvement in the formation of this peculiar disulfide bridge.…”

“…In spite of these differences in half‐cystine pairings, these toxins still fold according to the α/β scaffold, which is assumed to represent a structural prerequisite for ion channel blocking activity. The maintenance of the α/β motif is, however, accompanied by small structural modifications, such as the relative orientation between the axis of the helix and the β‐sheet structures, that impact the pharmacological affinity and/or selectivity toward their ion channel targets8–10. These variations in pharmacological profiles resulting from rearrangements of disulfide bridges are likely due to the spatial repositioning of some key amino acid residues that are involved in ion channel pore recognition.…”

Structural determinants of protein folding

“…In this case, the disulfide-pairing arrangement and consequent conformation of maurotoxin, a 34-mer potassium channel blocker isolated from scorpion venom, was shown to be drastically influenced by the presence of proline residues [85]. Despite possessing a conserved cysteine framework of the four-disulfide-bridged scorpion toxin, maurotoxin adopts a novel cysteine-pairing pattern (C1-C5, C2-C6, C3-C4, and C7-C8) that is distinct from the classical cysteine-pairing framework (C1-C5, C2-C6, C3-C7, and C4-C8) seen in Pi1 and HsTx1 toxins, the other two members of the same family.…”