The structure of the complex of calmodulin with KAR-2 -: A novel mode of binding explains the unique pharmacology of the drug
The structure of the complex of calmodulin with KAR-2 -: A novel mode of binding explains the unique pharmacology of the drug
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DOI：
10.1074/jbc.m410353200
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发表时间：
2005-03-04
影响因子：
4.8
通讯作者：
Ovádi, J
中科院分区：
文献类型：
--
作者：
Horváth, I;Harmat, V;Ovádi, J
3'-（β -氯乙基）-2',4'-二氧基-3,5'-螺-恶唑烷-4-去乙酰氧基长春碱（卡尔-2）是一种有效的抗微管药物，可以阻止癌细胞有丝分裂而没有明显的毒副作用。在这项研究中，我们证明除了靶向微管外，KAR-2还可以结合钙调蛋白，从而对抗三氟拉嗪的拮抗作用。为了确定KAR-2的两种性质的基础，它与Ca2+钙调素配合物的三维结构已经在溶液中使用核磁共振和x射线衍射结晶时进行了表征。n -15标记的钙调素的异相关（H-1-N-15异核单量子相干）光谱表明该蛋白与KAR-2结合后发生了全局构象变化（关闭）。2.12埃分辨率下的晶体结构显示出更完整的图像；KAR-2与钙调蛋白N端和C端氨基酸残基形成的新结构结合。虽然首先通过x射线衍射检测到结晶的三元配合物，但这种构象变化与核磁共振光谱表征的溶液结构一致。值得注意的是，当钙调素与KAR-2结合时，形成了与三氟拉嗪类似的三级复合物，尽管这两种配体（在大多数情况下）接触的是不同的氨基酸残基。这些观察结果解释了KAR-2作为抗微管药物的特异性；该药物与钙调蛋白上的一个新的药物结合域相互作用。因此，KAR-2不能阻止钙调素结合其大多数生理靶点。
3'-(beta- Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca2+-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation (H-1-N-15 heteronuclear single quantum coherence) spectra of N-15-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-Angstrom resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C- terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.