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Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination 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 Zn2+-free and Zn2+-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.

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Figure 1: Rpn11 and Rpn8 form a heterodimer through two distinct interfaces.
Figure 2: Rpn11 is missing a conserved binding site for the Ile44 patch of ubiquitin.
Figure 3: Missing proximal contacts allow Rpn11 cleavage promiscuity.
Figure 4: The Ins-1 loop of Rpn11 acts as a flap to fold over the ubiquitin C terminus.
Figure 5: Model for Rpn11-mediated deubiquitination.

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We thank the members of the Martin laboratory for helpful discussions and D. Morgan (University of California, San Francisco) for providing expression constructs for E1 and E2 enzymes. We are also grateful to M. Herzik (University of California, Berkeley) and A. Lyubimov (Stanford University) for help with crystallography data collection and processing. E.J.W. acknowledges support from the US National Science Foundation Graduate Research Fellowship. This research was funded in part by the Searle Scholars Program (A.M.), start-up funds from the University of California, Berkeley Department of Molecular and Cell Biology (A.M.) and the US National Science Foundation CAREER Program (NSF-MCB-1150288 to A.M.).

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Authors and Affiliations



E.J.W. and A.M. designed experiments; E.J.W. and C.P. expressed, purified and characterized protein variants; and E.J.W. performed X-ray crystallographic studies. E.J.W. and A.M. prepared the manuscript.

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Correspondence to Andreas Martin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Electron density of the Rpn11 catalytic active site in the apo and Zn2+-bound states.

(a) The 2|Fo|-|Fc| electron density map of the Zn2+-bound active site is shown in grey and contoured at 1.5σ. Depicted in cyan is the |Fo|-|Fc| difference electron density map contoured at 5σ, calculated from the final coordinates after three rounds of ADP and reciprocal space XYZ refinement with the Zn2+ ion and the catalytic water omitted. (b) 2|Fo|-|Fc| electron density map of the Rpn11 active site in the apo state, contoured at 1.5σ. Also included is the corresponding |Fo|-|Fc| difference electron density map contoured at 5σ, which does not show any peaks at this contouring level.

Supplementary Figure 2 Specific interface residues facilitate Rpn11–Rpn8 heterodimer formation.

Close-up view of interactions between Rpn11 (green) and Rpn8 (blue, surface representation) at interface 2. Rpn8 Gly170 packs against Rpn11 Leu34, Leu35, and Leu38 (shown in stick representation). For the interaction of the corresponding residue Rpn11 Met212 with Rpn8 see Figure 1e.

Supplementary Figure 3 Alignment of the hydrophobic dimerization interfaces of Rpn11 and related MPN-containing proteins.

Residues of the hydrophobic interfaces 1 and 2 predicted to contribute >0.45 kcal/mol of binding energy between Rpn11 and Rpn8 are colored red. Residues predicted to be at the same position in other proteins are colored red if hydrophobic.

Supplementary Figure 4 Alignment of the hydrophobic dimerization interfaces of Rpn8 and related MPN-containing proteins.

Residues of the hydrophobic interfaces 1 and 2 predicted to contribute >0.45 kcal/mol of binding energy between Rpn11 and Rpn8 are colored red. Residues predicted to be at the same position in other proteins are colored red if hydrophobic.

Supplementary Figure 5 Structure-based alignment of Rpn11 and AMSH-LP.

Supplementary Figure 6 Example gels for the Lys48-linked diubiquitin cleavage assay of Rpn11.

Shown are the gels for one Michealis-Menten experiment analyzing the 30-minute time courses of Lys48-linked di-ubiquitin cleavage by wild-type Rpn11-Rpn8 (5 μM) at substrate concentrations between 15 and 500 μM. Bands indicated by asterisks are due to contaminating proteins that co-purify at low abundance with Rpn11 and Rpn8 heterodimers. Covalently linked ClpX hexamer was used for normalization of staining and enzyme concentrations across different gels, which were all processed in parallel.

Supplementary Figure 7 The N terminus and Ins-2 region of Rpn11 contact Rpn2.

Crystal structures of the Rpn11-Rpn8 dimer and Rpn2 (ref. 1) (PDB ID: 4ADY), with Rpn11 in green, Rpn8 in blue and Rpn2 in orange, are docked into the segmented EM 3D-reconstruction of the substrate-bound 26S proteasome2 (EMDB ID: EMD-5669). The Ins-1 loop of Rpn11 is colored gold. The N-terminal residue of Rpn11 as well as the residues flanking its unstructured Ins-2 region are colored red and labeled. Extra electron density not accounted for by the crystal structures of Rpn11-Rpn8 and Rpn2, and presumably corresponding to the Ins-2 region of Rpn11, is circled.

Supplementary Figure 8 Fluorescence-based assays for Rpn11 ubiquitin binding and cleavage.

(a) Example time-based polarization measurements of the cleavage of 5 and 10 μM Ub-Lys-TAMRA by 1.25 μM Rpn11-Rpn8. (b) Example single-turnover kinetics measurement for the cleavage of 100 nM Ub-Lys-TAMRA by 450 μM Rpn11-Rpn8. The data are fit by a single exponential, with a calculated kcat = 0.95 min−1. (c) Michaelis-Menten curve for Ub-Lys-TAMRA cleavage by Rpn11, where kcat was constrained to the experimentally determined value in (b). Limitations in substrate solubility precluded using Ub-Lys-TAMRA at concentrations higher than KM, so measurement of a complete curve was not possible. The estimated KM for Ub-Lys-TAMRA cleavage by Rpn11 is 20 μM. (d) Tryptophan fluorescence-based assay of K48-linked di-ubiquitin binding to Rpn11V80A. Tryptophan fluorescence of 5 μM Rpn11-Rpn8 heterodimers was measured in the presence of Lys48-linked di-ubiquitin at concentrations between 0 and 500 μM, as discussed in the methods. Triplicate fluorescence measurements were averaged and fit to a simple binding curve with a KD of 67 μM.

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Worden, E., Padovani, C. & Martin, A. Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation. Nat Struct Mol Biol 21, 220–227 (2014).

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