Volume 24, Issue 8, Pages 1335-1345 (August 2016) Molecular Understanding of USP7 Substrate Recognition and C-Terminal Activation  Lionel Rougé, Travis W. Bainbridge, Michael Kwok, Raymond Tong, Paola Di Lello, Ingrid E. Wertz, Till Maurer, James A. Ernst, Jeremy Murray  Structure  Volume 24, Issue 8, Pages 1335-1345 (August 2016) DOI: 10.1016/j.str.2016.05.020 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1335-1345DOI: (10.1016/j.str.2016.05.020) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 C-Terminal Peptide of USP7 Restores Full Ubiquitin Cleavage Activity to the Catalytic Domain of USP7 and Regulates Enzyme Activity (A) Schematic of the USP7 domain structure and boundaries. (B) Sequence alignment of the C-terminal 19 residues of USP7 orthologs. (C) Enzyme kinetics for USP7CD-CTP constructs of varying linker lengths. See also Table S1 and Figure S1B. Structure 2016 24, 1335-1345DOI: (10.1016/j.str.2016.05.020) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Structure of USP7CD-UBL45 Covalently Bound to Ubiquitin (A) Cartoon and surface representation of the complex between USP7CD-UBL45 covalently bound to ubiquitin showing trans activation of the C-terminal peptide from an NCS-related molecule. One chain is represented as a light blue molecular surface and the other chain as a violet ribbon; ubiquitin is wheat colored. The catalytic cysteine is colored green and the side chains of Ile1100 and Ile1098 from one protomer are shown as spheres. Only one dimer from the dimer of dimers present in the asymmetric unit is shown. (B) Close up of C-terminal peptide binding in the activation cleft. The catalytic domain is represented as a cartoon colored dark green. Hydrogen bond interactions involving the C-terminal peptide are shown as black dashes. See also Figure S2 for details of the C-terminal peptide interaction in the USP7CD-CTP structure. (C) 2|Fo| − |Fc| electron density for the C-terminal peptide contoured at 1 σ. (D) The effect of addition of unlabeled ubiquitin onto 2H/13C/15N-labeled USP7CD. (E) The effect of addition of unlabeled ubiquitin onto 2H/13C/15N-labeled USP7CDGGS10-CTP. In both (D) and (E), a zoomed region of the 15N TROSY-HSQC spectra of USP7CD (pink on the left) and USP7CDGGS10-CTP (blue on the right) on their own and after addition of unlabeled native ubiquitin (from bovine erythrocytes; Sigma) in a 10-fold access (light blue and green) is shown. Arrows show the chemical shifts observed from the direct interaction. Asterisks mark the appearance of new signals in the USP7CDGGS10-CTP spectra. See also Figure S2. Structure 2016 24, 1335-1345DOI: (10.1016/j.str.2016.05.020) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Structure of the USP7CD-UBL123 (A) Cartoon diagram for USP7CD-123 with the catalytic domain colored orange and UBL1, UBL2, and UBL3 colored cyan, slate, and pink respectively. (B) Close up of the catalytic triad showing the residues are in the inactive state. (C) 2|Fo| − |Fc| electron density for the C-terminal α10 helix contoured at 1 σ. Structure 2016 24, 1335-1345DOI: (10.1016/j.str.2016.05.020) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 A Model for Full-Length USP7 in the Inactive Conformation and the Proposed Model for Full-Length USP7 in the Active Conformation (A) Schematic of the structures and their domain boundaries that were used to generate the proposed model for full-length USP7. (B) Structure of the UBL12345 domain (PDB: 4YOC) with UBL123 colored according to (A). (C) Model of USP7CD-UBL12345 generated by the superposition of the UBL123 domain of PDB: 4YOC on to the UBL123 domains of the USP7CD-UBL123 structure. (D) Proposed mechanism of cis activation of full-length USP7 by the C-terminal region. See also Figure S3A for a molecular model of full-length USP7 in complex with DNMT1 and Figure S3C for a molecular model of full-length USP7 consistent with trans activation by the C-terminal peptide. (E) Schematic model depicting activation of the catalytic domain by the C-terminal peptide in the presence of ubiquitin. Structure 2016 24, 1335-1345DOI: (10.1016/j.str.2016.05.020) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 MDM2 Binding by Full-Length USP7 (A) SPR sensorgrams showing binding to immobilized full-length MDM2 by (A) USP7-full length or (B) NTD-CD. (C) Superposition of sensorgrams for MDM2 binding by USP7-full length (red) USP7CD-UBL12345 (blue), negative protein control (green). (D) Superposition of sensorgrams for MDM2 binding by USP7-full length (red) and USP7ΔCTP (blue) constructs. Residuals are shown in insets for (A) and (B). (E) Binding constants for USP7, MDM2 interactions. MDM2 was immobilized at a target surface density of 1,000 RU. Analyte concentrations are 1,000 to 26 nM in 1.5-fold dilutions for USP7-FL and USP7ΔCTP or 2000–336 nM for USP7CD-UBL12345. For experiments in (C) and (D), USP7 was applied at a fixed concentration of 500 nM, and sensorgrams for association and dissociation phases are compared. Data shown are representative of multiple experiments. Structure 2016 24, 1335-1345DOI: (10.1016/j.str.2016.05.020) Copyright © 2016 Elsevier Ltd Terms and Conditions