Delineation of the functional site of α-dendrotoxin -: The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins
Delineation of the functional site of α-dendrotoxin -: The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins
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DOI：
10.1074/jbc.273.39.25393
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发表时间：
1998-09-25
影响因子：
4.8
通讯作者：
Ménez, A
中科院分区：
文献类型：
--
作者：
Gasparini, S;Danse, JM;Ménez, A
我们使用基于定点诱变和化学合成的突变方法，鉴定了对于 α-树突毒素 (α DTX) 与大鼠脑突触体膜上的 Kv1 钾通道结合至关重要的残基。其 59 个残基中的 26 个单独被丙氨酸取代。 Lys(5) 和 Leu(9) 的取代使亲和力降低超过 1000 倍，Arg(3)、Arg(4)、Leu(6) 和 Ile(8) 的取代使亲和力降低 5-30 倍。用正亮氨酸或鸟氨酸取代 Lys(5) 也极大地改变了 α DTX 的结合特性。与野生型 α DTX 相比，所有这些类似物都显示出相似的圆二色光谱，表明这些取代均不会影响毒素的整体构象。 Ser(38) 和 Arg(46) 的取代也降低了毒素的亲和力，但此外还改变了其二色性，表明这两个残基发挥了结构作用。其他残基被排除在识别位点之外，因为它们的取代没有引起显着的亲和力变化。因此，α DTX 的功能位点包括六个主要结合残基，全部位于其 N 末端区域，其中 Lys5 和 Leu(9) 是最重要的。将 α DTX 与另一种树毒素 DTX-K 的功能位点进行比较（Smith, L. A.、Reid, P. F.、Wang, F. C.、Parcej, D. N.、Schmidt, J. J.、Olson, M. A. 和 Dolly, J. O. (1997) Biochemistry 36, 7690-7696），表明它们仅共享主要功能位点赖氨酸和可能的亮氨酸残基；一种毒素的附加功能残基与另一种毒素不同。将 α DTX 的功能位点与来自有毒无脊椎动物的结构上不相关的钾通道阻断毒素的功能位点进行比较，发现共同存在突出的关键赖氨酸，具有紧密的重要疏水残基（Leu、Tyr 或 Phe）和少量其他残基。因此，无论其系统发育起源如何，所有这些毒素可能都经历了功能趋同。 α DTX 的功能位点在拓扑上与结构类似的牛胰腺胰蛋白酶抑制剂的“抗蛋白酶位点”无关。
We identified the residues that are important for the binding of alpha-dendrotoxin (alpha DTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys(5) and Leu(9) decreased affinity more than 1000-fold, and substitutions of Arg(3), Arg(4), Leu(6), and Ile(8) by 5-30-fold. Substitution of Lys(5) by norleucine or ornithine also greatly altered the binding properties of alpha DTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type alpha DTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser(38) and Arg(46) also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Thus, the functional site of alpha DTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu(9) being the most important. Comparison of the functional site of alpha DTX with that of DTX-K, another dendrotoxin (Smith, L. A., Reid, P. F., Wang, F. C., Parcej, D. N., Schmidt, J. J., Olson, M. A, and Dolly, J. O. (1997) Biochemistry 36, 7690-7696), reveals that they only share the predominant lysine and probably a leucine residue; the additional functional residues differ from one toxin to the other. Comparison of the functional site of alpha DTX with those of structurally unrelated potassium channel-blocking toxins from venomous invertebrates revealed the common presence of a protruding key lysine with a close important hydrophobic residue (Leu, Tyr, or Phe) and few additional residues. Therefore, irrespective of their phylogenetic origin, all of these toxins may have undergone a functional convergence. The functional site of alpha DTX is topographically unrelated to the "antiprotease site" of the structurally analogous bovine pancreatic trypsin inhibitor.