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^NTD) 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^NTD. 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^NTD and K48-diUb. In this structure, Rpn13^NTD 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^NTD similarly as the Ub monomer in the known Rpn13^NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13^NTD. Thus, a charge-reversal mutation in Rpn13^NTD 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^NTD 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很重要。仅表征了Rpn13（Rpn13 ^NTD）的N端结构域与Ub单体之间的复杂结构，而目前尚不清楚Rpn13如何特异性识别K48连接的Ub链。使用单分子FRET，在这里我们显示K48连接的双泛素（K48-diUb）在不同的构象状态之间波动，并且Rpn13 ^NTD选择性地丰富了先前存在的紧密状态。对于全长Rpn13和更长的K48连接的Ub链，观察到相同的结合模式。使用溶液NMR光谱，我们确定了Rpn13 ^NTD和K48-diUb之间的复杂结构。在此结构中，Rpn13^NTD同时与保持在复合物中的K48-diUb的近端和远端Ub亚基相互作用，从而证实了smFRET的发现。与已知Rpn13 ^NTD：Ub结构中的Ub单体相似，近端Ub与Rpn13 ^NTD相互作用，而远端Ub与Rpn13 ^NTD的大部分静电表面结合。因此，Rpn13 ^NTD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，从而导致泛素化蛋白的积累。此外，远端Ub进入Rpn13 ^NTD的物理障碍具有邻近连接的Ub单体的化合物可能会破坏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的连接选择性提供了结构基础，从而为调节蛋白酶体功能打开了新的窗口。