• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions.
【24h】

Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions.

机译：重构KRAB-KAP-1阻遏物复合物：一个模型系统，用于定义RING-B盒-螺旋-螺旋结构域介导的蛋白质-蛋白质相互作用的分子解剖。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins. KRAB-mediated gene silencing requires binding to the corepressor KAP-1. The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface. Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity. We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components. Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain. A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain. The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions. KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies. Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity. Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding. Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain. The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition. These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins. Copyright 2000 Academic Press.
机译：KRAB结构域是真核锌指蛋白中常见的75个氨基酸残基的转录抑制模块。 KRAB介导的基因沉默需要与共抑制因子KAP-1结合。 KRAB：KAP-1相互作用需要KAP-1的RING-B盒螺旋线圈（RBCC）域，该域是分布广泛的基序，被认为是蛋白质-蛋白质界面。关于RBCC介导的配体结合以及单个亚域在识别和特异性中的作用知之甚少。我们已经通过使用纯化的成分重建和表征KRAB：KAP-1-RBCC相互作用来解决这些问题。我们的结果表明，与KAP-1结合的KRAB是直接且特异性的，因为来自TIF1alpha和MID1的相关RBCC域不结合KRAB域。凝胶过滤，分析超速离心，化学交联，非变性凝胶电泳和定点诱变技术的结合显示，KAP-1-RBCC必须低聚为同源三聚体才能结合KRAB结构域。 KAP-1-RBCC和KRAB结合的寡聚化需要RING指，B2框和卷曲螺旋区域，因为这些域中的突变会同时消除这些功能。通过化学交联和速度沉降研究发现，KRAB域结合可稳定KAP-1-RBCC的同聚状态。突变的KAP-1-RBCC分子与野生型KAP-1杂合，但这些复合物对于KRAB结合无效，表明潜在的显性负活性。用异源二聚化，三聚化或四聚化结构域取代卷曲螺旋区域无法概括KRAB结构域结合。包含RING，RING-B盒或MID1的卷曲螺旋区域的嵌合KAP-1-RBCC蛋白也无法结合KRAB结构域。 KAP-1-RBCC介导与KRAB结构域的高度特异性，直接相互作用，并且似乎起着集成的，可能的合作结构单元的作用，其中每个子结构域均有助于寡聚和/或配体识别。这些观察结果为RBCC域介导的蛋白质-蛋白质相互作用提供了第一个原理，并且对于鉴定RBCC域蛋白质的新配体具有启示。版权所有2000学术出版社。

著录项

相似文献

  • 外文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号