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

Abstract

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.

Ubiquitin signaling in cell cycle control and tumorigenesis

“…Dysregulation of the ubiquitination modification led to uncontrolled cell cycle progression and eventually resulted in tumorigenesis [18]. Based upon this notion, targeting the ubiquitin system has provided effective therapeutic strategies for cancer treatment [129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145][146]. At the moment, how these signaling events are integrated and orchestrated in a time-space-dependent manner remains not fully understood.…”

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties

“…Groups of mice (n = 3) were injected intra peritoneally (i.p.) with a single dose of RA375 (5,10,20,40,60, 100 mg/Kg in 25% β-hydroxypropyl cyclodextrin) and endpoints evaluated included clinical observation and body weight for one week. No adverse effects were noted with RA375 at even the highest dose.…”

“…Two ubiquitin receptors, RPN13 and RPN10 in the 19S RP act in concert to recognize protein substrates tagged with K48-linked chains of �4 ubiquitin subunits. In addition to its ubiquitin binding Pru N-terminal domain [5], RPN13 also binds the deubiquitinase (DUB) UCH37/UCHL5, and RPN2 recruiting it to the 19S RP [6,7]. RPN13 interaction also enhances the DUB activity of UCH37, likely by opening up its active site [8,9].…”

The Length of a Ubiquitin Chain: A General Factor for Selective Recognition by Ubiquitin‐Binding Proteins

“…phosphorylation and acetylation), the length of the Ub chains adds another layer of complexity to the Ub code. Up to now, it is not known how the Ub chain length may affect the interaction with UBPs and thus Ub signaling, as there is only limited information on the mode of interaction of UBPs with long Ub chains in general and the potential preference for a certain chain length in particular …”