Gasparini S, Danse J M, Lecoq A, Pinkasfeld S, Zinn-Justin S, Young L C, de Medeiros C C, Rowan E G, Harvey A L, Ménez A
Département d'Ingéniérie et d'Etudes des Protéines, CEA Saclay, 91191 Gif sur Yvette Cedex, France.
J Biol Chem. 1998 Sep 25;273(39):25393-403. doi: 10.1074/jbc.273.39.25393.
We identified the residues that are important for the binding of alpha-dendrotoxin (alphaDTX) 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 Lys5 and Leu9 decreased affinity more than 1000-fold, and substitutions of Arg3, Arg4, Leu6, and Ile8 by 5-30-fold. Substitution of Lys5 by norleucine or ornithine also greatly altered the binding properties of alphaDTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type alphaDTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser38 and Arg46 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 alphaDTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu9 being the most important. Comparison of the functional site of alphaDTX 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 alphaDTX 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 alphaDTX is topographically unrelated to the "antiprotease site" of the structurally analogous bovine pancreatic trypsin inhibitor.
我们采用基于定点诱变和化学合成的突变方法，确定了大鼠脑突触体膜上α-树突毒素（αDTX）与Kv1钾通道结合的重要残基。其59个残基中的26个分别被丙氨酸取代。Lys5和Leu9的取代使亲和力降低了1000倍以上，而Arg3、Arg4、Leu6和Ile8的取代使亲和力降低了5至30倍。用正亮氨酸或鸟氨酸取代Lys5也极大地改变了αDTX的结合特性。与野生型αDTX相比，所有这些类似物都显示出相似的圆二色光谱，表明这些取代都不会影响毒素的整体构象。Ser38和Arg46的取代也降低了毒素的亲和力，但此外还改变了其二色性特性，表明这两个残基起结构作用。其他残基被排除在识别位点之外，因为它们的取代不会引起显著的亲和力变化。因此，αDTX的功能位点包括六个主要结合残基，均位于其N端区域，其中Lys5和Leu9最为重要。将αDTX的功能位点与另一种树突毒素DTX-K（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）的功能位点进行比较，发现它们仅共享主要的赖氨酸残基，可能还有一个亮氨酸残基；其他功能残基因毒素而异。将αDTX的功能位点与来自有毒无脊椎动物的结构不相关的钾通道阻断毒素的功能位点进行比较，发现普遍存在一个突出的关键赖氨酸以及一个与之相邻的重要疏水残基（Leu、Tyr或Phe）和少数其他残基。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。αDTX的功能位点在拓扑结构上与结构类似的牛胰蛋白酶抑制剂的“抗蛋白酶位点”无关。
