Structure of Proteasome Ubiquitin Receptor hRpn13 and Its Activation by the Scaffolding Protein hRpn2  Xiang Chen, Byung-Hoon Lee, Daniel Finley, Kylie J. Walters  Molecular Cell  Volume 38, Issue 3, Pages 404-415 (May 2010) DOI: 10.1016/j.molcel.2010.04.019 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 38, 404-415DOI: (10.1016/j.molcel.2010.04.019) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Structure of hRpn13's Uch37-Binding Domain (A) Primary and secondary structure of proteasome component hRpn13 highlighting its Pru domain in blue and its C-terminal structural domain in orange. Regions of low sequence complexity (as determined by the program seg; Wootton and Federhen, 1996) are indicated with an “X” and experimentally determined β strands and α helices with arrows and cylinders, respectively. (B) hRpn13's C-terminal domain forms a helical bundle. A ribbon diagram is displayed highlighting Rpn13's nine α helices, with the orientation of structure on the left rotated by 90° relative to that on the right. This figure and that in (C) displays only one possible orientation for H1, which is not defined relative to H2-H9. Nt, N terminus; Ct, C terminus. (C) Surface and ribbon view of hRpn13 displaying hydrophobic, basic, and acidic residues involved in binding to Uch37 in green, blue, and red, respectively, as defined by intermolecular NOE data (Figure S1D) and amino acid substitutions (Figure 1E). (D) Sequence of Uch37 spanning Lys300–Lys329, with the hRpn13-binding surface (313–329) (Hamazaki et al., 2006) highlighted in yellow and its polar residues in red. (E) GST pull-down assays to assess binding of His-Uch37 to GST-tagged wild-type (WT) or amino acid-substituted (lanes 1–4, defined in right panel) hRpn13 (253–407) loaded onto glutathione S-Sepharose resin. GST-tagged hRpn13 (1–150) and direct loading of His-Uch37 were used as negative and positive controls, respectively. Blotting was done with anti-His (top) or anti-GST (bottom) antibody. See also Figure S1. Molecular Cell 2010 38, 404-415DOI: (10.1016/j.molcel.2010.04.019) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 hRpn13's Pru Domain Interacts with Its C-Terminal Region Spanning Ser253–Asp407 (A) NOE interactions identified between hRpn13's Pru domain and its C-terminal region. Each panel contains a selected region of an 15N-dispersed NOESY experiment recorded on 15N, 50% 2H-labeled hRpn13 full-length protein. Interdomain NOEs are labeled in red. (B) 1H, 15N HSQC spectrum of hRpn13 with Cys88 adducted to MTSL (red) reveals amide protons with broadened resonances due to the close proximity of the spin label. A control experiment with MTSL quenched by ascorbic acid is also displayed (black). The enlarged regions highlight inter- (top) and intra- (bottom) domain interactions. (C and D) Systematic MTSL spin labeling experiments like that shown in (B) were performed with labeling at six different residue positions, as displayed in (C). In each case, only one cysteine was present, and all native cysteines were substituted with alanine. In (D), paramagnetic relaxation enhancement data is summarized according to Equation (1) for each labeling scheme.(1) 1−IparaIdia (E) The data in (D) is summarized onto a ribbon diagram with hRpn13's Pru and C-terminal domains displayed in blue and orange, respectively. The PRE distance constraints are highlighted with dashed lines. The color coding in (D) and (E) follows that defined in (C). See also Table S1 and Figure S2. Molecular Cell 2010 38, 404-415DOI: (10.1016/j.molcel.2010.04.019) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Structure of Full-Length hRpn13 (A) NMR-derived structure of hRpn13 displaying the Pru and C-terminal domains in blue and orange, respectively. Thirty-one structures are displayed, with residues in secondary structure elements superimposed. Randomly coiled regions, including the 21 N- and C-terminal residues and the linker between the Pru domain and H1, are omitted for clarity. (B) 15N hetNOE data reveal high-frequency motions for the N and C termini and the linker regions neighboring helix H1. Averaged values for the various structural regions are displayed for ready comparison. Error bars were determined from a repeated control spectrum, as described in Experimental Procedures. (C) NMR data from two different sites of contact between the Pru domain and C-terminal region are highlighted. NOE interactions unique to the full-length protein are noted in the expanded red box, whereas the expanded blue box highlights interdomain PRE interactions observed between S1a, the S1a-S1b loop, and S1b of hRpn13's Pru domain and MTSL-labeled Thr332Cys. See also Figure S3. Molecular Cell 2010 38, 404-415DOI: (10.1016/j.molcel.2010.04.019) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 hRpn13's Ubiquitin-Binding Activity Is Activated by Proteasome Scaffolding Protein hRpn2 (A) Ribbon and surface display of an hRpn13:ubiquitin model structure highlighting steric clashes (in red) that occur if the interdomain interactions are preserved. hRpn13's Pru domain and C-terminal region are shown in blue and orange, respectively, with its ubiquitin-, hRpn2-, and Uch37-binding surfaces in yellow, purple and green, respectively. Ubiquitin is in gray. This model demonstrates why ubiquitin binding is facilitated when hRpn2 abrogates interdomain interactions. (B and C) Intrinsic tryptophan quenching was used to determine the affinity between hRpn13 full-length protein and ubiquitin alone and with 10-fold molar excess of an hRpn2 fragment spanning 797–953. The data were compared to hRpn13 (1–150), which encompasses its Pru domain. (D) hRpn2 (797–953) and ubiquitin bind to opposite surfaces of hRpn13's Pru domain. The hRpn13 amino acids within the hRpn2 (797–953) binding surface, as determined by an NMR titration experiment, are highlighted in purple on a ribbon diagram of hRpn13's Pru domain. The ubiquitin-binding loops are displayed in yellow. (E) Superimposed 1H, 15N HSQC spectra of 15N-labeled hRpn13 with Cys88 (left) or Thr332Cys (right) adducted to MTSL without (blue) or with equimolar quantities of unlabeled hRpn2 (797–953) (red). Control experiments prior to MTSL treatment and hRpn2 addition (black) and in which MTSL effects are quenched with ascorbic acid after hRpn2 addition (green) are included to reveal that Lys34, Ile270, and Ala272 are affected by interdomain distance-dependent paramagnetic relaxation enhancements only in the absence of hRpn2. See also Figure S4. Molecular Cell 2010 38, 404-415DOI: (10.1016/j.molcel.2010.04.019) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Assembly into the Proteasome Potentiates Ubiquitin Binding by hRpn13 (A) Composition of human proteasomes purified from either red blood cells (RBC) or from an Rpn11-tagged line of HEK293 cells (Wang et al., 2007a). 1.5 μg of proteasomes were analyzed by SDS-PAGE and immunoblotting. The asterisk indicates an ∼32 kDa putative isoform or cleaved form of S5a. See Figure S5 for further characterization of these proteasomes. (B) hRpn13 targets ubiquitinated cyclin B (polyUb-cyclin B) for degradation by purified proteasomes. hRpn13 (WT or Phe76Arg variant) was added to RBC proteasomes at 20- or 40-fold molar excess, as indicated. After a 5 min preincubation, reactions were initiated by the addition of substrate. Reactions were quenched by the addition of SDS-PAGE sample buffer and were subjected to SDS-PAGE/immunoblot analysis, using antibodies to cyclin B. Proteasome was present in all reactions at 4 nM. (C and D) Experiments were performed as described in (B) but with hRpn13 full-length protein (WT), hRpn13 (1–150), or hRpn13 (253–407). The results were quantified and plotted in (D). (E) Model of hRpn13 docked into the proteasome. Rpn13's interdomain interactions are abrogated as it binds to the proteasome's scaffolding protein hRpn2, thus priming it for ubiquitinated substrates and Uch37. We propose that the long flexible linker region following the Pru domain may facilitate progressive cleavage of ubiquitin moieties from substrates. See also Figure S5. Molecular Cell 2010 38, 404-415DOI: (10.1016/j.molcel.2010.04.019) Copyright © 2010 Elsevier Inc. Terms and Conditions