1. Volume 26, Issue 1, Pages 72-84.e7 (January 2018)
Selectively Modulating Conformational States of USP7 Catalytic Domain for Activation 
Ayşegül Özen, Lionel Rougé, Charlene Bashore, Brian R. Hearn, Nicholas J. Skelton, Erin C. Dueber 
Structure 
Volume 26, Issue 1, Pages e7 (January 2018)
DOI: /j.str
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2. Structure 2018 26, 72-84.e7DOI: (10.1016/j.str.2017.11.010)
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3. Figure 1 Comparative Analysis of Inactive and Active USP7 States
(A) Active and inactive states of USP7cd were previously captured in crystal structures; the catalytic triad geometry is disrupted in the apo crystal structure (PDB: 1NB8) while ubiquitin-aldehyde conjugated USP7cd structures reveal canonical cysteine protease active-site geometry (PDB: 1NBF).
(B) Three distance metrics from a molecular dynamics trajectory show a transition from the inactive to active state starting from the inactive state with protonated catalytic H464 and deprotonated H294 and C223 (left). In the inactive state (PDB: 1NB8, gray cartoon), H294, likely protonated, participates in an electrostatic network involving W285, E298, and Y224, which is disrupted in the active state (PDB: 1NBF, green cartoon).
(C) Crystal structures of USP2cd and USP7cd in the active and the inactive states. The densely packed phenylalanine cluster in USP2cd and corresponding residues in USP7cd are depicted in space-filling representation. In the active USP7cd, core packing by smaller hydrophobics (mostly leucines) is suboptimal, leaving a small, open groove, whereas in the inactive state a denser packing of these residues negates the groove.
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4. Figure 2 RosettaDesign Consensus Sequences
RosettaDesign predicts that Y224F, L299A, and V302K mutations likely stabilize the active state but not the inactive state.
(A) The low-energy sequences optimized for the positions 224, 260, 279, 288, 294, 299, and 302 based on two protomers in the wild-type active-state crystal structure (PDB: 1NBF, chains A [top] and B [bottom]).
(B) The low-energy sequences optimized based on two protomers in the wild-type inactive-state crystal coordinates (PDB: 1NB8, chains A [top] and B [bottom]; see Figure S3 for the third protomer in 1NBF that is in the inactive state, chain E).
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5. Figure 3 Kinetic Analysis of Designs
Michaelis-Menten parameters (A) kcat/KM, (B) kcat, and (C) KM for wild-type and four designs in the backgrounds of USP7cd and USP7cd-45. Error bars represent SDs from at least three replicates (see Figure S5 and Table S4).
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6. Figure 4 Cleavage of Tetrameric Ubiquitin Chains
The activity of the designs on tetrameric ubiquitin chain substrates was assessed by gel cleavage assays for the wild-type (WT) USP7cd-45 and USP7cd constructs and the four USP7cd single mutants Y224F, H294E, L299A, and V302K.
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7. Figure 5 Switching Loop Interactions and Core Packing of USP7 Variants
Detailed views of the (A) Ub∼USP7cd WT, (B) apo USP7cd WT, (C) Ub∼USP7cd L299A, (D) Ub∼USP7cd H294E protomer A, (E) Ub∼USP7cd H294E protomer B, (F) Ub∼USP7cd V302K with bound malonic acid, (G) Ub∼USP7cd V302K, and (H) apo USP7cd V302K protomer A; and (I) protomer B. Key residues are shown as sticks or spheres (i.e., core-packing residues).
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8. Figure 6 Molecular Interactions across the Ub∼USP7cd Interface
Detailed view of ubiquitin-USP7cd interactions in the (A) wild-type, (WT) (B and C) H294E (chains A and B), (D) L299A, and (E and F) V302K (with and without a bound malonic acid) complex crystal structures. Wild-type USP7cd is colored green, designs teal, and ubiquitin yellow. The side chains that surround the hydrophobic grooves F283, L299, and V302 are represented by spheres. The ubiquitin residues that make charge interactions with USP7cd and key USP7cd residues, including the K302 side chain in V302K complex structures, are shown as sticks.
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9. Figure 7
Switching Loop Conformations in Designs Compete with C-Terminal Tail Activation
The potential clashes between the USP7cd C-terminal tail and the switching loop are assessed by comparing the recently published ternary C-terminal tail-bound Ub∼USP7cd structure with the crystal structures of (A) the wild-type (WT), (B) the H294E/B, (C) the L299A, and (D) the V302K complexes. The clashes are shown in red. C-terminal tail overlaid onto the Ub∼USP7cd complex structures of the designs reveals clashes at predominantly two hydrophobic sites I1098 and I1100 that are critical for activation by the C-terminal tail. The complexes are in surface representation with ubiquitin colored yellow, with the wild-type and mutant USP7cd colored green and teal, respectively. The switching loop residues 283–298 are highlighted in purple.
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10. Structure 2018 26, 72-84.e7DOI: (10.1016/j.str.2017.11.010)
Copyright © 2017 Elsevier Ltd Terms and Conditions