Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation

• Published in: Nature structural & molecular biology Vol. 21; no. 3; pp. 220 - 227
• Main Authors: Worden, Evan J, Padovani, Chris, Martin, Andreas
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-03-2014; Nature Publishing Group
• Subjects: 631/337/474/582; 631/535; Amino Acid Sequence; Biochemistry; Biological Microscopy; Crystallography, X-Ray; Crystals; Endopeptidases - chemistry; Endopeptidases - genetics; Escherichia coli - metabolism; Kinetics; Life Sciences; Membrane Biology; Molecular Sequence Data; Mutation; Proteases; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - genetics; Protein Multimerization; Protein Structure; Protein Structure, Tertiary; Proteins; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Sequence Homology, Amino Acid; Structure; Substrates; Ubiquitin; Ubiquitin - chemistry; Zinc - chemistry; United States
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb.2771

Rpn11, the only essential deubiquitinase (DUB) of the 26S proteasome, sits at the top of the substrate entry pathway and facilitates substrate degradation through cotranslocational deubiquitination. The structure of the Rpn11–Rpn8 complex, together with functional assays, offers insight into Rpn11's promiscuous DUB activity during proteasomal degradation.
Polyubiquitin chains target protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 to allow efficient substrate degradation. Despite Rpn11's essential function during substrate processing, its detailed structural and biochemical characterization has been hindered by difficulties in purifying the isolated enzyme. Here we report the 2.0-Å crystal structures of Zn
2+
-free and Zn
2+
-bound
Saccharomyces cerevisiae
Rpn11 in an MPN-domain heterodimer with Rpn8. The Rpn11-Rpn8 interaction occurs via two distinct interfaces that may be conserved in related MPN-domain complexes. Our structural and mutational studies reveal that Rpn11 lacks a conserved surface to bind the ubiquitin Ile44 patch, does not interact with the moiety on the proximal side of the scissile isopeptide bond and exhibits no linkage specificity for ubiquitin cleavage. These findings explain how Rpn11 functions as a promiscuous deubiquitinase for cotranslocational substrate deubiquitination during proteasomal degradation.