Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin
Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin
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DOI：
10.1016/j.abb.2016.02.014
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发表时间：
2016-03-15
影响因子：
3.9
通讯作者：
Nikawa, Takeshi
中科院分区：
文献类型：
--
作者：
Ohno, Ayako;Ochi, Arisa;Nikawa, Takeshi
Cbl-b是一种RING型泛素连接酶。以前，我们发现Cbl-b介导的IRS-1的泛素化和蛋白体降解有助于由卸载应力引起的肌肉萎缩。磷酸化五肽DGpYMP（Cblin）模拟Tyr 612磷酸化IRS-1并在体外和体内抑制Cbl-b介导的IRS-1的泛素化和降解。在这项研究中，我们使用NMR证实了Cblin与Cbl-b的TKB结构域之间的直接相互作用。此外，我们发现缩短的三肽GpYM也结合到TKB结构域。为了阐明Cblin的抑制机制，我们以2.5埃的分辨率解析了TKB-Cblin复合物的晶体结构。Cblin中的pY通过氢键网络和疏水相互作用插入TKB结构域中的带正电荷的口袋中。在该复合物中，Cblin结构非常类似于另一种底物衍生的磷酸肽Zap-70衍生的磷酸肽的TKB结合形式。这些肽缺乏pY和参与TKB结构域结合的保守残基之间的保守肽内氢键。代替保守的相互作用，这些肽特异性地与TKB结构域相互作用。基于Cblin与TKB结构域的这种结合模式，我们可以设计针对卸载介导的肌肉萎缩的药物。(C)2016 Elsevier Inc. All rights reserved.
Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 angstrom. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy. (C) 2016 Elsevier Inc. All rights reserved.