Liu Zhu, Dong Xu, Yi Hua-Wei, Yang Ju, Gong Zhou, Wang Yi, Liu Kan, Zhang Wei-Ping, Tang Chun
1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071 China.
2National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070 China.
Cell Discov. 2019 Apr 2;5:19. doi: 10.1038/s41421-019-0089-7. eCollection 2019.
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 (Rpn13) 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 Rpn13. 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 Rpn13 and K48-diUb. In this structure, Rpn13 simultaneously 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 Rpn13 similarly as the Ub monomer in the known Rpn13:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13. Thus, a charge-reversal mutation in Rpn13 weakens 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 Rpn13 with 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.
K48连接的泛素（Ub）链与Rpn13之间的相互作用对于泛素化底物蛋白的蛋白酶体降解至关重要。目前仅对Rpn13的N端结构域（Rpn13）与Ub单体之间的复合结构进行了表征，而Rpn13如何特异性识别K48连接的Ub链仍不清楚。在此，我们利用单分子荧光共振能量转移表明，K48连接的双泛素（K48-diUb）在不同构象状态之间波动，并且一种预先存在的紧密状态被Rpn13选择性富集。全长Rpn13与更长的K48连接的Ub链也观察到相同的结合模式。利用溶液核磁共振光谱，我们确定了Rpn13与K48-diUb之间的复合结构。在该结构中，Rpn13同时与K48-diUb的近端和远端Ub亚基相互作用，这两个亚基在复合物中保持结合状态，从而证实了单分子荧光共振能量转移的结果。近端Ub与Rpn13的相互作用类似于已知Rpn13:Ub结构中的Ub单体，而远端Ub则结合到Rpn13的一个主要为静电的表面。因此，Rpn13中的电荷反转突变会削弱Rpn13与K48连接的Ub链之间的相互作用，导致泛素化蛋白积累。此外，用邻近连接的Ub单体物理阻断远端Ub与Rpn13的接触会破坏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连接选择性提供了结构基础，这为调节蛋白酶体功能打开了一扇新窗口。
