CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common α/β scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 µg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat Kv1.2 (Kd value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 µM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat Kv1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on Kv channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.
- chemical synthesis
- cobatoxin 1
- docking simulation
- K+ channel
- scorpion toxin
- three-dimensional structure
↵1 These authors contributed equally to this work.
Abbreviations used: CoTX1, cobatoxin 1; ACoTX1, synthetic structural cobatoxin 1 analogue with Pro and Gln at positions 7 and 9, respectively; NOE, nuclear Overhauser effect; DQF-COSY, double-quantum-filtered correlation spectroscopy; DIANA, distance geometry algorithm for NMR; CNS, crystallography and NMR system; HN, amide proton; Fmoc, Nα-(9-fluorenyl)methyloxycarbonyl; SKCa channel, small-conductance Ca2+-activated K+ channel; IKCa1 channel, intermediate-conductance Ca2+-activated K+ channel; hIKCa1, human IKCa1; Kv channel, mammalian voltage-gated K+ channel; Shaker B channel, insect voltage-gated K+ channel; Shaker B Δ44 channel, Shaker B channel formed by α subunits truncated at their N-terminal extremity (region 1–44); TFA, trifluoroacetic acid; MALDI-TOF, matrix-assisted laser-desorption ionization–time-of-flight; 3-D, three-dimensional; r.m.s.d., root mean square deviation.
- The Biochemical Society, London ©2004