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首页> 外文期刊>Journal of Molecular Biology >The buried diversity of bovine seminal ribonuclease: Shape and cytotoxicity of the swapped non-covalent form of the enzyme
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The buried diversity of bovine seminal ribonuclease: Shape and cytotoxicity of the swapped non-covalent form of the enzyme

机译：牛精原核糖核酸酶的掩埋多样性：交换的非共价形式酶的形状和细胞毒性

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摘要

Bovine seminal ribonuclease exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein. Surprisingly, the cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer. This buried diversity comes into light in the reducing environment of the cytosol, where the unswapped dimer dissociates into monomers, whereas the swapped one generates a metastable dimeric form (NCD-BS) with a quaternary assembly that allows the molecule to escape the protein inhibitor of ribonucleases. The stability of this quaternary shape was mainly attributed to the combined presence of Pro19 and Leu28. We have prepared and fully characterized by X-ray diffraction the double mutant P19A/L28Q (PALQ) of the seminal enzyme. While the swapped and unswapped forms of the mutant have structures very similar to that of the corresponding wild-type forms, the non-covalent form (NCD-PALQ) adopts an opened quaternary structure, different from that of NCD-BS. Moreover, model building clearly indicates that NCD-PALQ can be easily sequestered by the protein inhibitor. In agreement with these results, cytotoxic assays have revealed that PALQ has limited activity, whereas the single mutants P19A and L28Q display cytotoxic activity against malignant cells almost as large as the wild-type enzyme. The significant increase in the antitumor activity, brought about by the substitution of just two residues in going from the double mutant to the wild-type enzyme, suggests a new strategy to improve this important biological property by strengthening the interface that stabilizes the quaternary structure of NCD-BS. (C) 2007 Elsevier Ltd. All rights reserved.
机译：牛精原核糖核酸酶以交换和未交换的二聚体的平衡混合物形式存在于天然状态。两种异构体的分子包膜和暴露表面几乎没有区别，它们的多样性几乎完全掩埋在蛋白质内部。令人惊讶地，该酶的细胞毒性和抗肿瘤活性是交换的二聚体的独特性质。这种被掩盖的多样性在细胞质的还原环境中得以显现，其中未交换的二聚体解离成单体，而被交换的二聚体产生具有亚四级组装的亚稳二聚体形式（NCD-BS），从而使该分子能够逃脱蛋白抑制剂的抑制。核糖核酸酶。该四元形状的稳定性主要归因于Pro19和Leu28的组合存在。我们已经准备并通过X射线衍射对精酶的双突变体P19A / L28Q（PALQ）进行了充分表征。尽管突变体的交换形式和非交换形式具有与相应的野生型形式非常相似的结构，但非共价形式（NCD-PALQ）采用的开放性四级结构与NCD-BS不同。此外，模型建立清楚地表明，NCD-PALQ可以很容易地被蛋白质抑制剂隔离。与这些结果一致，细胞毒性试验表明，PALQ的活性有限，而单个突变体P19A和L28Q对几乎与野生型酶一样大的恶性细胞显示出细胞毒活性。从双突变体到野生型酶中仅两个残基的取代引起的抗肿瘤活性的显着提高，提出了一种新的策略，可通过加强稳定四价结构的界面来改善这一重要的生物学特性。 NCD-BS。 （C）2007 Elsevier Ltd.保留所有权利。

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