Fig 2: Docking models of the K48 di-ubiquitin (Ub):UCH37•RPN13DEUBAD complex.(A–B) HADDOCK docking models show two poses corresponding to the interaction between K48 di-Ub and UCH37 along with their fit to experimental small-angle X-ray scattering data of the K48 di-Ub:UCH37C88A•RPN13DEUBAD complex. The goodness of fit to the experimental intensity is represented by ?2 values. In the first pose (A), the proximal Ub (Ubprox; green) interacts with a5–6 of UCH37. In the second pose (B), the distal Ub (Ubdist; orange) interacts with a5–6 of UCH37. (C) Residues highlighting the interaction between the aromatic-rich region of UCH37 a5–6 and the I44 patch of Ubprox in pose 1 (A). (D) Residues highlighting the interaction between the aromatic-rich region of UCH37 a5–6 and the I44 patch of Ubdist in pose 2 (B). (E) Polar contacts between Ubprox and UCH37 a5–6 in pose 1 (A). (F) Contacts between Ubdist and residues of UCH37 located outside the a5–6 motif in pose 1 (A). (G) Contacts between Ubprox and residues of UCH37 located outside the a5–6 motif in pose 2 (B). (H) The relative location of active site and the scissile, K48 isopeptide bond in molecular docking poses 1 and 2. In pose 1, residues of a6 and the loop leading into the catalytic Cys (C88) form a barrier for the isopeptide bond. In pose 2, only Q82 of UCH37 blocks the K48 isopeptide bond.

Fig 3: Proposed debranching model.(A) Surface depiction of UCH37 showing the canonical S1 (cS1) ubiquitin (Ub)-binding site and the new K48-specific binding sites identified in this study. (B) Proposed mechanism for chain debranching using the K48-specific binding sites. The K48-linked portion of a branched chain engages the K48-specific Ub-binding sites in two different orientations: one with the proximal Ub (Ubprox) docked at the a5–6 motif and the other with the distal Ub (Ubdist) bound to that site. As the docking models show (Figure 4H), the K48 isopeptide bond is less obstructed and closer to the catalytic C88 residue when the K48 Ubdist moiety is bound to a5–6 and Ubprox is bound near the L181 region. In this orientation, the other Ubdist at the branchpoint is positioned near the frontside of the enzyme. With K48 isopeptide bond cleavage occurring on the backside, the a5–6 motif would serve as the noncanonical S1 (ncS1) site and the L181 region would be the ncS1´ site.

Fig 4: The catalytic domain (CD) of UCH37 confers K48 linkage specificity.(A) Structure of UCH37 bound RPN13DEUBAD (PDB ID: 4UEL; see also 4WLR) highlighting residues comprising the canonical S1 (cS1) Ubiquitin (Ub)-binding site (shown in pink). (B) Original hypothesis for how UCH37 uses the cS1 site along with an unknown S1' site to catalyze K48 chain debranching. (B) SDS-PAGE analysis of 6×His-UCH37C88A-mediated pulldowns with M1-, K6-, K11-, K29-, K33-, K48-, and K63-linked Ub chains. Each chain (50 pmol) was mixed with 6×His-UCH37C88A (5 nmol) immobilized on Ni-NTA resin. (C) SDS-PAGE analysis of 6×His-UCH37(CD)C88A with M1-, K6-, K11-, K29-, K33-, K48-, and K63-linked Ub chains. Each chain (50 pmol) was mixed with 6×His-UCH37(CD)C88A (5 nmol) immobilized on Ni-NTA resin. (D–E) Isothermal titration calorimetry analysis of UCH37(CD)C88A binding to K48 tri-Ub (C), and K63 tri-Ub. (D) (F) Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) analysis of the interaction between UCH37(CD)C88A and K48 tri-Ub. * denotes a UV peak without corresponding light scattering data. (G) Theoretical and calculated molar mass of complexes detected by SEC-MALS. Figure 1—source data 1.ITC and SEC-MALs analysis of UCH37C88A and UCH37C88A (CD) with Ub chains.

Fig 5: Identifying proteins dependent on the K48 chain binding and debranching activity of UCH37 for degradation.(A) Scheme for tandem mass tagging (TMT)-based proteomics of UCH37KO cells expressing either wild-type (WT) UCH37, UCH37I216E, or UCH37F117A. (B–C) Volcano plots comparing the proteomes of UCH37F117A- and WT UCH37-expressing UCH37KO cells (B), and UCH37I216E- and WT UCH37-expressing UCH37KO cells. (C) In both cases, the cells are treated with H2O2 to induce oxidative stress. (D) Biological process analysis of proteins upregulated in UCH37F117A- and UCH37I216E-expressing UCH37KO cells. (E) Western blot analysis of the turnover of POLR2D and mitofusin-2 (MFN2) after shutting off translation with emetine in WT and UCH37KO HEK293 cells. (F) Western blot analysis of the turnover of POLR2D and MFN2 after shutting off translation with emetine in UCH37KO cells reconstituted with either UCH37F117A or UCH37I216E. Immunoblotting with a-POLR2D, a-MFN2, and a-actin antibodies. (G) Scheme showing the enrichment and analysis K6-linked ubiquitin chains. (H) Western blot analysis of K6-affimer-enriched ubiquitinated species from WT and UCH37KO HEK293 cells. The enriched conjugates were treated with OTUB1 (1 µM) or UCH37•RPN13 (2 µM) at rt for 1 hr. Immunoblotting with a-MFN2 and a-K48-linkage specific antibodies. Figure 7—source data 1.Tandem mass tagging proteomics analysis of WT, UCH37KO and UCH37 mutants (F117A or I216E) transduced cells. Figure 7—source data 2.Uncropped gel images of Western blot analysis of emetine chase and K6-affimer enrichment pulldown assay.