Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction

Abstract

Targeted protein degradation is largely performed by the ubiquitin-proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a 1 , which is not essential for ubiquitin-mediated protein degradation in budding yeast 2 . In the accompanying manuscript we report that Rpn13 3-7 , a component of the 9-subunit proteasome base, functions as a ubiquitin receptor 8 , complementing its known role in docking deubiquitinating enzyme Uch37/UCHL5 [4][5][6] to the proteasome. Here, we merge crystallography and NMR data to describe Rpn13's ubiquitin binding mechanism. We determined the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for Rpn13's role as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13's role as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37. The structure of murine Rpn13 (mRpn13) (1-150) was determined at 1.7 Å resolution by Xray crystallography, and found to contain a Pleckstrin Homology (PH) domain fold ( Figure 1a and 1b) (Structure determination and refinement statistics are provided in the Supplement). In particular, whereas the first 21 N-and last 20 C-terminal amino acids are unstructured, residues 22-130 form a PH domain fold. This result was surprising, as primary sequence alignment did not identify Rpn13 to be homologous to previously characterized proteins. This finding coupled with its ubiquitin receptor properties 8 prompted us to name the N-terminal domain of Rpn13 Pleckstrin-like receptor for ubiquitin (Pru).Though very divergent at their sequence level, all PH domains have a common β-sandwich fold. The PH domain of Rpn13 is composed of a 4-stranded twisted antiparallel β-sheet (β 1-4 : residues 22-34, 45-52, 56-62, 71-74) that packs almost orthogonally against a second triple stranded β-sheet (β 5-7 : residues 80-85, 92-98, 103-110) (Supplementary Figure 1). Similar to other PH domains, Rpn13 Pru forms a hydrophobic core containing conserved hydrophobic residues (F26, V47, I49, F59, F82, Y94, L96, F107 and M109), which are located within β-sheets. One end of the β-sandwich is capped by a long Cterminal amphipathic α-helix (residues 117-128), which is stabilized by interactions between V124 and L128, whereas the other corner of the hydrophobic core is closed by three loops formed by residues located between strands S1/S2, S3/S4 and S6/S7 ( Figure 1a and Supplementary Figure 1).Despite much effort, we were unable to crystallize the Rpn13 Pru:ubiquitin complex; however, we were able to...

The E3 Ubiquitin Ligase Parkin Is Recruited to the 26 S Proteasome via the Proteasomal Ubiquitin Receptor Rpn13

“…We have previously characterized the Pru domain at the N terminus of Rpn13 as a novel ubiquitin-binding domain, establishing Rpn13 as one of the major Ub receptors within the proteasome (Fig. 1A) (35,36). By using 1:1 interaction studies in yeast (Figs.…”

“…The proteasomal ubiquitin receptors Rpn10 (34) and Rpn13/ ADRM1 (35,36) can directly bind ubiquitinated cargo at the 19 S proteasome (see Fig. 1A).…”

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

“…Another shuttling factor, the p62/sequestosome1, associates with the 26S proteasome through the N-terminal domain (Seibenhener et al, 2004;Myeku and Figueiredo-Pereira, 2011), and participate in the selective recognition and degradation of ubiquitinated cargo by selective autophagy (Lamark et al, 2009). While Rpn1 is responsible for binding shuttling factors such as Rad23 and Dsk2 (Elsasser et al, 2002(Elsasser et al, , 2004Rosenzweig et al, 2012), and the non-obligatory deubiquitinating enzyme Ubp6/Usp14 Elsasser et al, 2004;Rosenzweig et al, 2012), Rpn2 only binds the Rpn13 subunit (Schreiner et al, 2008;Rosenzweig et al, 2012). This specific binding and the delivery of ubiquitinated proteins to the proteasome by shuttling factors is driven by the Nterminal ubiquitin-like domain (UBL), while the C-terminal ubiquitin associated domain (UBA) is responsible for ubiquitin binding (Rosenzweig et al, 2012).…”

“…Rpn10 binds ubiquitin conjugates through its C-terminus Ubiquitin-Interacting Motif (UIM), and a similar function is mediated by the Rpn13 subunit through a conserved amino-terminal region named pleckstrin-like receptor for ubiquitin (Pru) domain Schreiner et al, 2008;Sakata et al, 2012). The Pru domain is also important to attach Rpn13 to the proteasome (Schreiner et al, 2008).…”

“…The interaction of polyubiquitinated proteins with the RP base occurs on two intrinsic proteins, Rpn10 and Rpn13, which are apically located in order to better capture ubiquitinated substrates (Sakata et al, 2012). Rpn10 binds ubiquitin conjugates through its C-terminus Ubiquitin-Interacting Motif (UIM), and a similar function is mediated by the Rpn13 subunit through a conserved amino-terminal region named pleckstrin-like receptor for ubiquitin (Pru) domain Schreiner et al, 2008;Sakata et al, 2012). The Pru domain is also important to attach Rpn13 to the proteasome (Schreiner et al, 2008).…”