• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Novel isoform-specific interfaces revealed by PKA RIIbeta holoenzyme structures.
【24h】

Novel isoform-specific interfaces revealed by PKA RIIbeta holoenzyme structures.

机译：由PKA RIIbeta全酶结构揭示的新型同工型特异性界面。

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 cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike RI subunits, RII subunits are both substrates and inhibitors. Because RIIbeta knockout mice have important disease phenotypes, the RIIbeta holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain, although this linker determines the unique globular architecture of the RIIbeta holoenzyme. To understand how RIIbeta functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RIIbeta constructs. In the absence of nucleotide, RIIbeta(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RIIbeta(108-268) holoenzyme structure (1.62 A) with AMP-PNP/Mn(2+) showed that we trapped the RIIbeta subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn(2+) may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIbeta(102-265), which contained six additional linker residues. The additional linker residues in RIIbeta(102-265) increased the affinity to 1.6 nM, suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure, this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the alphaF-alphaG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the RIIbeta holoenzyme.
机译：cAMP依赖性蛋白激酶催化（C）亚基受到两类功能性非冗余调节（R）亚基RI和RII的抑制。与RI亚基不同，RII亚基既是底物又是抑制剂。因为RIIbeta基因敲除小鼠具有重要的疾病表型，所以RIIbeta全酶是开发同工型特异性激动剂和/或拮抗剂的靶标。我们也几乎不知道将抑制剂位点连接至N端二聚结构域的接头区域，尽管该接头决定了RIIbeta全酶的独特球状结构。为了解RIIbeta如何同时充当抑制剂和底物并阐明接头的结构作用，我们设计了不同的RIIbeta构建体。在缺少核苷酸的情况下，包含单个环状核苷酸结合结构域的RIIbeta（108-268）结合C亚基的能力较弱，而对于AMP-PNP（一种不可水解的ATP类似物），亲和力为11 nM。具有AMP-PNP / Mn（2+）的RIIbeta（108-268）全酶结构（1.62 A）显示，我们将RIIbeta亚基困在具有C亚基的封闭结构构象的酶：底物复合物中。 AMP-PNP / Mn（2+）提供的增强的亲和力可能是增加亲和力和捕获其他蛋白底物与其同源蛋白激酶的有用策略。因为诱变预测抑制剂位点N末端的区域可能与RI和RII停靠的位置不同，所以我们还设计了RIIbeta（102-265），其中包含6个额外的接头残基。 RIIbeta（102-265）中的其他接头残基将亲和力提高至1.6 nM，表明对接至该表面也可能提高催化效率。在相应的全酶结构中，该接头以延伸链的形式对接在大叶的表面上。由αF-αG环形成并在许多蛋白激酶中保守的疏水口袋也为PKI的两亲性螺旋提供了停靠位点。接头肽的这种新颖方向为该区域如何促进RIIbeta全酶的独特组织提供了第一个线索。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 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