蠍毒中鉀離子通道抑制蛋白結構之探討

Abstract

Tcl是從學名為Tityus cambridgei的蠍子毒液中所分離出的鉀離子通道抑制勝?，全長由23個胺基酸(ACGSC-RKKCK-GSGKC-INGRC-KCY)所組成並含有3對雙硫鍵，是目前在蠍毒中所發現最小的鉀離子通道抑制勝?，可辨識Shaker B及膜電位敏感型(voltage-gated)鉀離子通道並抑制其活性；我們藉由固相勝?合成技術製得並摺疊成具活性組態的Tcl，以逆相高效液相層析管柱純化後，進一步利用旋光光譜儀及核磁共振光譜儀解出Tcl的三級結構，同時定出其雙硫鍵的相對聯結關係為Cys^ 2-Cys^ 15、Cys ^5-Cys^ 20及Cys^ 9-Cys^ 22，而藉由X-PLOR分析軟體模擬計算可知Tcl的N端Ser^ 4-Lys^ 10形成螺旋及3^ 10螺旋結構，在Gly^ 13-Ile^ 16及Arg^ 19-Tyr^ 23則具有一段反平行的β摺疊，利用計算所得最佳的15個最小能量結構的重疊，可得其骨幹(backbone)的均方根偏差值(root mean squaro deviation)為0.26±0.05?；根據與其他結構及功能性相關的蠍毒三級結構上的比較，我們認為具有較長胺基酸組成的蠍毒中，其某些胺基酸的存在對鉀離子通道的抑制作用是非必要的，而蠍毒中抑制膜電位敏感型鉀離子通道活性的重要區段及胺基酸則主要位於C端；因此藉由Tcl三級結構的研究跟探討，除了提供一些有價值的資訊幫助對離子通道的認識外，同時也希望對新藥物的設計與開發可提供實質上的參考價值。

Tel is a new K(superscript +) channel-blocking peptide identified from the scorpion venom of Tityus cambridgei and composed of 23 amino acid residues with three disulfide bridges. It is the shortest known toxin from scorpion venom that recognizes the Shaker B K(superscript +) channels and the voltage-gated K(superscript +) channels in brain. Synthetic Tel was produced by solid-phase synthesis and purified by reversed-phase HPLC. The pairings of three disulfide bridges in the synthetic Tel were identified as Cys^ 2-Cys^ 15、Cys^ 5-Cys^ 20、Cys^ 9-Cys^ 22 by NMR experiments. The NMR solution structures of Tel were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tel contains an α-helix and a 310 helix at N-terminal Ser4-Lys10 and a double-stranded β-sheet at Gly13-Ile16 and Arg19-Tyr23, with a type I?β turn at Asn17-Gly18. Superposition of each structure with the best structure yielded an average root mean square deviation (RMSD) of 0.26±0.05? for the backbone atoms in residues 2 to 23. The 3D structure of Tcl was compared to two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the activity of the scorpion K+ channel blockers and found that some of the residues in the larger scorpion K(superscript +) channel blockers (31?40 amino acids) are not directly involved in K(superscript +) channel blocking activity. The structure study of Tcl might shed light on understanding the mechanism of the inhibitory activity by the channel blocker. Furthermore, it may also provide more information for designing new lead compounds to control the dysfunctions of K(superscript +) channels.