ROLE OF TYROSINE AND TRYPTOPHAN RESIDUES IN THE STRUCTURE ACTIVITY RELATIONSHIPS OF A CARDIOTOXIN FROM NAJA-NIGRICOLLIS VENOM
ROLE OF TYROSINE AND TRYPTOPHAN RESIDUES IN THE STRUCTURE ACTIVITY RELATIONSHIPS OF A CARDIOTOXIN FROM NAJA-NIGRICOLLIS VENOM
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DOI：
10.1021/bi00399a004
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发表时间：
1987-12-15
期刊：
影响因子：
2.9
通讯作者：
MENEZ, A
中科院分区：
文献类型：
--
作者：
GATINEAU, E;TOMA, F;MENEZ, A
本文试图定位蛇心脏毒素毒性的关键区域。毒素.gamma。是一种由 60 个氨基酸组成的单链多肽，从非洲喷眼镜蛇 (Naja nigricollis) 的毒液中分离出来。三个芳香族残基，即Trp-11、Tyr-22和Tyr-51，已通过化学方法单独进行修饰。通过圆二色性、荧光、质子磁共振光谱和两种特异性单克隆抗体仔细研究了天然毒素和每种衍生物的结构。所有化学修饰都不会改变毒素的整体结构，在所有情况下，毒素都保持折叠成三个相邻的环（I、II和III），这些环富含从含有四个二硫桥的小球状区域出现的β-折叠片。然而，与天然毒素的结构相比，在每种衍生物的结构中都检测到了许多细微的变化。特别是，Tyr-51 的硝化引起了球状区域的结构扰动。 Tyr-22 的硝化引起分子的 β-折叠区域发生更实质性的变化。因此，在 Tyr-22 和 Tyr-51 之间的天然毒素中观察到的强环间 NOE 在 Tyr-22 衍生物中消失，并且在球状区域中观察到显着变化。相反，在Trp-11修饰后没有检测到环II和III的β-折叠结构的改变。对该衍生物观察到的所有变化仍然位于环 I 中 Trp-11 吲哚侧链附近。测量了修饰的生物学后果：小鼠体内的致死效力和体外对 FL 细胞的细胞毒性活性。致死活性与细胞毒性相关：Tyr-51 修饰毒素与天然毒素具有同等效力，而 Tyr-22 和 Trp-11 衍生毒素的特点是活性显着降低，Trp-11 衍生毒素的效力最低。我们得出的结论是：(1) Tyr-51 不参与毒素的功能位点，尽管它与分子核心相互作用；(2) Tyr-22 可能发挥双重结构和功能作用；(3) Trp-11 位于或接近毒素的功能位点。这些数据表明环 I 在确定心脏毒素毒性方面的重要性。
This paper is an attempt to localize the critical area determining toxicity in a snake cardiotoxin. Toxin .gamma. is a single-chain polypeptide of 60 amino acids, which has been isolated from the venom of the African spitting cobra, Naja nigricollis. Three aromatic residues, namely, Trp-11, Tyr-22, and Tyr-51, have been individually modified by chemical means. The structure of the native toxin and of each derivative has been carefully investigated by circular dichroism, fluorescence, proton magnetic resonance spectroscopy, and two specific monoclonal antibodies. None of the chemical modifications alters the overall structure of the toxin, which in all cases remains folded into three adjacent loops (I, II, and III) rich in .beta.-pleated sheet emerging from a small globular region containing four disulfide bridges. A number of subtle changes, however, have been detected in the structure of each derivative compared with that of the native toxin. In particular, nitration of Tyr-51 provoked a structural perturbation in the globular region. Nitration of Tyr-22 induces a more substantial change in the .beta.-sheet area of the molecule. Thus, the strong inter-ring NOE that is observed in the native toxin between Tyr-22 and Tyr-51 vanishes in the Tyr-22 derivative, and significant changes are observed in the globular region. In contrast, no alteration of the .beta.-sheet structure of loops II and III has been detected after modification of Trp-11. All changes observed for this derivative remain located in the vicinity of the indole side chain of Trp-11 in loop I. The biological consequences of the modifications were measured: the lethal potency in vivo in mice and the cytotoxic activities in vitro on FL-cells. Lethal activities correlate with cytotoxicity: Tyr-51 modified toxin is equally potent as native toxin, whereas Tyr-22 and Trp-11 derivatized toxins are characterized by substantially lesser activities, the Trp-11 derivatized toxin being the least potent. We conclude that (1) Tyr-51 is not involved in the functional site of the toxin, although it is in interaction with the core of the molecule, (2) Tyr-22 may play a dual structural and functional role and (3) Trp-11 is in, or in close proximity to, the functional site of the toxin. These data indicate the importance of loop I in determining toxicity of the cardiotoxin.