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首页> 外文期刊>Journal of Molecular Biology >NMR Reveals the Interplay among the AMSH SH3 Binding Motif, STAM2, and Lys63-Linked Diubiquitin
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NMR Reveals the Interplay among the AMSH SH3 Binding Motif, STAM2, and Lys63-Linked Diubiquitin

机译：NMR揭示了AMSH SH3结合基序，STAM2和与Lys63连接的双泛素之间的相互作用

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摘要

AMSH [associated molecule with a Src homology 3 domain of signal transducing adaptor molecule (STAM)] is one of the deubiquitinating enzymes associated in the regulation of endocytic cargo trafficking. It shows an exquisite selectivity for Lys63-linked polyubiquitin chains that are the main chains involved in cargo sorting. The first step requires the ESCRT-0 complex that comprises the STAM and hepatocyte growth factor-regulated substrate (Hrs) proteins. Previous studies have shown that the presence of the STAM protein increases the efficiency of Lys63-linked polyubiquitin chain cleavage by AMSH, one of the deubiquitinating enzyme involved in lysosomal degradation. In the present study, we are seeking to understand if a particular structural organization among these three key players is responsible for the stimulation of the catalytic activity of AMSH. To address this question, we first monitored the interaction between the ubiquitin interacting motif (UIM)-SH3 construct of STAM2 and the Lys63-linked diubiquitin (Lys63-Ub2) chains by means of NMR. We show that Lys63-Ub2 is able to bind either the UIM or the SH3 domain without any selectivity. We further demonstrate that the SH3 binding motif (SBM) of AMSH (AMSH-SBM) outcompetes Lys63-Ub2 for binding SH3. Additionally, we show how different AMSH-SBM variants, modified by their sequence and length, exhibit similar equilibrium dissociation constants when binding SH3 but significantly differ in their dissociation rate constants. Finally, we report the solution NMR structure of the AMSH-SBM/SH3 complex and propose a structural organization where the AMSH-SBM interacts with the STAM2-SH3 domain and contributes to the correct positioning of AMSH prior to polyubiquitin chains' cleavage. (C) 2016 Elsevier Ltd. All rights reserved.
机译：AMSH [信号转导衔接子分子（STAM）具有Src同源性3结构域的相关分子]是与内吞货物运输调控相关的去泛素化酶之一。它显示了Lys63连接的多聚泛素链的精湛选择性，后者是货物分拣的主要链。第一步需要包含STAM和肝细胞生长因子调节的底物（Hrs）蛋白的ESCRT-0复合物。先前的研究表明，STAM蛋白的存在可提高AMSH裂解Lys63连接的多泛素链的效率，AMSH是参与溶酶体降解的一种去泛素化酶。在本研究中，我们试图了解这三个关键参与者中的特定结构组织是否负责刺激AMSH的催化活性。为了解决这个问题，我们首先通过NMR监测了STAM2的泛素相互作用基序（UIM）-SH3构建体与Lys63连接的双泛素（Lys63-Ub2）链之间的相互作用。我们显示，Lys63-Ub2能够结合UIM或SH3域而没有任何选择性。我们进一步证明，AMSH（AMSH-SBM）的SH3结合基序（SBM）优于Lys63-Ub2结合SH3。此外，我们显示了不同的AMSH-SBM变体（经其序列和长度修饰）如何在与SH3结合时表现出相似的平衡解离常数，但其解离速率常数却存在显着差异。最后，我们报告了AMSH-SBM / SH3复合物的溶液NMR结构，并提出了一个结构组织，其中AMSH-SBM与STAM2-SH3结构域相互作用，并有助于多泛素链切割之前AMSH的正确定位。 （C）2016 Elsevier Ltd.保留所有权利。

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