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. 2022 Apr 22;11:e76100. doi: 10.7554/eLife.76100

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© 2022, Du et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 1. — (A) Structure of UCH37 bound RPN13^DEUBAD (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-UCH37^C88A-mediated pulldowns with M1-, K6-, K11-, K29-, K33-, K48-, and K63-linked Ub chains. Each chain (50 pmol) was mixed with 6×His-UCH37^C88A (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 UCH37^C88A and UCH37^C88A (CD) with Ub chains.

elife-76100-fig1-data1.xlsx^{(972.6KB, xlsx)}

Figure 1—figure supplement 1. — (A) Structure of UCH37 bound RPN13^DEUBAD (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-UCH37^C88A-mediated pulldowns with M1-, K6-, K11-, K29-, K33-, K48-, and K63-linked Ub chains. Each chain (50 pmol) was mixed with 6×His-UCH37^C88A (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 UCH37^C88A and UCH37^C88A (CD) with Ub chains.

elife-76100-fig1-data1.xlsx^{(972.6KB, xlsx)}