Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 Reveal Distinct Binding Mechanisms between Substrate Receptors and Shuttle Factors of the Proteasome

Abstract

SUMMARY Three receptors (Rpn1/S2/PSMD2, Rpn10/S5a, Rpn13/Adrm1) in the proteasome bind substrates by interacting with conjugated ubiquitin chains and/or shuttle factors (Rad23/HR23, Dsk2/PLIC/ubiquilin, Ddi1) that carry ubiquitinated substrates to proteasomes. We solved the structure of two such receptors with their preferred shuttle factor, namely hRpn13Pru:hPLIC2UBL and scRpn1 T1:scRad23UBL. We find that ubiquitin folds in Rad23 and Dsk2 are fine-tuned by residue substitutions to achieve high affinity for Rpn1 and Rpn13 respectively. A single substitution in hPLIC2 yields enhanced interactions with the Rpn13 ubiquitin contact surface and sterically blocks hRpn13 binding to its preferred ubiquitin chain type, K48 diubiquitin. Rpn1 T1 binds two ubiquitins in tandem and we find that Rad23 binds exclusively to the higher affinity Helix28/Helix30 site. Rad23 contacts at Helix28/Helix30 are optimized compared to ubiquitin by multiple conservative amino acid substitutions. Thus, shuttle factors deliver substrates to proteasomes through fine-tuned ubiquitin-like surfaces.

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment

“…In SPS1 the density was well defined (Figure 3), indicating a relatively stable interaction with the 26S complex that may in part correspond to bound UBL domain proteins (Aufderheide et al, 2015; Bashore et al, 2015). The density contacted Rpn1 (Figure 3, Figure 4D, E), consistent with the bound UBL domains of Rad23 or the ubiquitin C-terminal hydrolase 6 (Ubp6)/USP14 (Chen et al, 2016b; Shi et al, 2016), and extended to another binding site of Rad23 at the Rpn10 subunit (Hiyama et al, 1999; Mueller and Feigon, 2003; Walters et al, 2002) (Figure 4D, F). Interestingly, similar to our SPS1 class, Ubp6-bound proteasomes have been shown to mainly adopt the s2 conformation (Aufderheide et al, 2015).…”

“…Poly-GA-associated proteasomes in substrate processing states showed additional densities that may correspond to bound ubiquitin and/or UBL domain-containing cofactors such as the deubiquitinating enzyme Ubp6/USP14 or the substrate shuttle factor Rad23 (Aufderheide et al, 2015; Bashore et al, 2015; Chen et al, 2016b; Shi et al, 2016). Although the binding of these factors to poly-GA-associated proteasomes remains to be conclusively demonstrated, several UBL domain proteins (Rad23, ubiquilin2 or Bag6) were highly enriched in the poly-GA interactome (May et al, 2014), and Rad23 is an important regulator of poly-GA induced toxicity (Zhang et al, 2016).…”

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

“…In addition, these same methyl residues displayed chemical shift changes upon Rpn1 association with Rad23 UBL (Figure 5D). A structure of Rad23UBL bound to residues in the same region was independently observed (Chen et al, 2016), confirming that Rpn1 PC1 likely contains overlapping (or partially overlapping) binding sites for polyUb and for Rad23 UBL .…”

Meddling with Fate: The Proteasomal Deubiquitinating Enzymes

“…Indirect recognition is mediated by a family of proteins often referred to as shuttling ubiquitin receptors. These shuttle proteins have an N-terminal ubiquitin-like (UBL) domain [18], which targets them to Rpn10, Rpn13, or Rpn1 [16,17,19–21], and a C-terminal ubiquitin-associated (UBA) domain, which binds ubiquitin conjugates [22]. Thus, before proteins are deubiquitinated at the proteasome, a prior step of ubiquitin binding may occur where many distinct interactions between ubiquitin and the proteasome-associated receptors are possible.…”