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Protein Phosphatase-1 Complex Disassembly by p97 is Initiated through Multivalent Recognition of Catalytic and Regulatory Subunits by the p97 SEP-domain Adapters
Journal of Molecular Biology ( IF 4.7 ) Pub Date : 2020-10-12 , DOI: 10.1016/j.jmb.2020.10.001
Matthias Kracht , Johannes van den Boom , Jonas Seiler , Alexander Kröning , Farnusch Kaschani , Markus Kaiser , Hemmo Meyer

The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.



中文翻译:

通过p97 SEP域适配器通过催化和调节亚基的多价识别来启动p97蛋白磷酸酶1复合物的拆卸。

AAA-ATPase VCP / p97与SEP域适配器p37,UBXN2A和p47协同作用,从蛋白磷酸酶1(PP1)剥离抑制剂3(I3)进行活化。与p97介导的降解过程相反,PP1复合物的分解是不依赖泛素的。因此,尚不清楚如何实现选择性靶向。使用生化重构和交联质谱,我们在这里显示SEP域适配器使用多价底物识别策略。预计会与SEP结构域一起形成螺旋的N末端序列元件与p97中心孔中的直接靶标I3结合并结合,从而展开,而其伴侣PP1被SHP框和UBX结构域之间的连接子保持锁定到p97的外围N域上。尽管I3结合元件在p47中起作用,体外p47需要从p37移植PP1结合接头才能进行活性强调,这两个位点对于控制特异性都是必不可少的。值得注意的是,展开随后受p47 N末端区域的抑制片段控制,提示其调节功能。总之,这项研究揭示了p97衔接子如何与蛋白质复合物结合,以实现不依赖泛素的分解,同时确保对一个亚基的选择性。

更新日期:2020-11-21
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