Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

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.