Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13
Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13
复制标题
蛋白酶体受体Rpn13识别K48连接的Ub链的结构基础
DOI：
10.1038/s41421-019-0089-7
复制
发表时间：
2019-04-02
期刊：
影响因子：
33.5
通讯作者：
Tang, Chun
中科院分区：
文献类型：
--
作者：
Liu, Zhu;Dong, Xu;Tang, Chun
k48连接的泛素（Ub）链和Rpn13之间的相互作用对于泛素化底物蛋白的蛋白酶体降解是重要的。目前只对Rpn13的n端结构域（Rpn13NTD）与Ub单体之间的复杂结构进行了表征，而Rpn13如何特异性识别k48连接的Ub链尚不清楚。利用单分子FRET，我们发现k48连接的二泛素（K48-diUb）在不同的构象状态之间波动，并且预先存在的致密状态被Rpn13NTD选择性地富集。在全长Rpn13和更长的k48连接的Ub链上观察到相同的结合模式。利用溶液核磁共振光谱测定了rpn13ntd与K48-diUb之间的复合物结构。在这个结构中，rpn13ntd同时与K48-diUb的近端和远端Ub亚基相互作用，这些亚基仍然与复合物相关，从而证实了smFRET的发现。近端Ub与Rpn13NTD的相互作用类似于已知Rpn13NTD:Ub结构中的Ub单体，而远端Ub与Rpn13NTD的静电表面结合。因此，rpn13ntd的电荷反转突变削弱了Rpn13与k48连接的Ub链之间的相互作用，导致泛素化蛋白的积累。此外，用邻近连接的Ub单体物理阻塞远端Ub与rpn13ntd的通路可以破坏Rpn13与K48-diUb之间的相互作用。综上所述，k48连接的Ub链与Rpn13的二价相互作用为Rpn13的连锁选择性提供了结构基础，为调节蛋白酶体功能打开了新的窗口。
The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, while it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have determined the complex structure between Rpn13NTDand K48-diUb. In this structure, Rpn13NTDsimultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTDsimilarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge-reversal mutation in Rpn13NTDweakens the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, physical blockage of the access of the distal Ub to Rpn13NTDwith a proximity-attached Ub monomer can disrupt the interaction between Rpn13 and K48-diUb. Taken together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.