Phosphorylation-induced conformational changes and functional regulation of the muscle-atrophy-associated ubiquitin ligase Cbl-b

为了阐明Cbl-b通过磷酸化增强泛素连接酶活性的结构基础，我们初步构建了Cbl-b^&lt；39-465&gt；Y363E突变体的蛋白表达系统，模拟Cbl-b的磷酸化状态。我们成功地开发了一种蛋白质纯化方案，以产生足够数量的纯蛋白质，用于X射线结晶学研究。我们对用该方法提纯的Cbl-b^&lt；39-465&Gt；Y363E进行了晶体筛选。然而，黄的团队在2012年1月报道了磷酸化的c-Cbl的晶体结构，c-Cbl是Cbl-b的同系物。因此，我们改变了原计划的研究项目，将重点放在Cbl-b^&lt；39-341&gt；(以下简称Cbl-bTKB)与磷酸化的IRS-1之间的相互作用上。我们成功地测定了Cbl-b TKB与磷酰化IRS-1的短肽模拟物的络合物的晶体结构。它揭示了Cbl-b TKB与磷酸肽结合方式的细节。

To elucidate the structural basis for enhancement of Cbl-b’s ubiquitin ligase activity through the phosphorylation, we initially constructed a protein expression system for Cbl-b^<39-465>Y363E mutant that mimics the phosphorylated state of Cbl-b. We succeed in the development of a protein purification protocol in order to generate pure protein in sufficient quantities for X-ray crystallographic studies. We carried out crystal screening of a Cbl-b^<39-465>Y363E purified by this protocol. However, Huang’s group reported the crystal structure of the phosphorylated c-Cbl, a homolog of Cbl-b, in January 2012. Thus, we changed the originally planned research project for the study focused on the interaction between the Cbl-b^<39-341>(hereafter called "Cbl-b TKB") and the phosphorylated IRS-1. We succeeded in the crystal structure determination of the Cbl-b TKB in complex with the short phosphopeptide mimetic of phospholylated IRS-1. It revealed the details of the binding mode of Cbl-b TKB to the phosphopeptide.