A role for intersubunit interactions in maintaining SAGA deubiquitinating module structure and activity

Abstract

The deubiquitinating module (DUBm) of the SAGA coactivator contains the Ubp8 isopeptidase, Sgf11, Sus1, and Sgf73, which form a highly interconnected complex. Although Ubp8 contains a canonical USP catalytic domain, it is only active when in complex with the other DUBm subunits. The Sgf11 zinc finger (Sgf11-ZnF) binds near the Ubp8 active site and is essential for full activity, suggesting that the Sgf11-ZnF helps maintain the active conformation of Ubp8. We report structural and solution studies showing that deletion of the Sgf11-ZnF destabilizes incorporation of Ubp8 within the DUBm, giving rise to domain swapping with a second complex and misaligning active site residues. Activating mutations in Ubp8 that partially restore activity in the absence of the Sgf11-ZnF promote the monomeric form of the DUBm. Our data suggest an unexpected role for Sgf11 in compensating for the absence of structural features that maintain the active conformation of Ubp8.

Figure 1. Gain-of-function mutations in Ubp8 that compensate for deletion of the Sgf11 zinc finger

(A) Structure of the wild-type DUBm. The four subunits are Ubp8 (green), Sgf11 (orange), Sus1 (blue), Sgf73 (salmon); zinc atoms are depicted as red spheres. The USP and ZnF-UBP domains of Ubp8 are indicated below. The Sgf11-ZnF (in dotted circle) binds to the USP domain of Ubp8 and is connected to its helical region by an extended linker. (B) The Sgf11-ZnF (orange) binds near the Ubp8 (green) active site, contacting loops L1 and L2. The catalytic residues, C146 and H427, and the oxyanion hole residue, N131 are depicted as sticks. Loop L1 (dashed gray line) is disordered in the absence of ubiquitin. (C) Rate of Ub-AMC cleavage as a function of substrate concentration by wild-type DUBm and DUBm lacking the Sgf11-ZnF containing either wild-type or Ubp8 with substitutions S144N, S149N, or S144N/S149. (D) Sequence alignment of USP DUBs indicated residues 144 and 149 of Ubp8. Diagram in green below sequence indicates secondary structure in Ubp8. (E) Superposition of Ubp8 (green) with hUSP8 (pink) showing hydrogen bonding between N149 in hUSP8 with loop L2 backbone residues. Residue S149 in Ubp8 does not contact loop L2. (F) Cleavage of K48-diubiquitin by wild-type and mutant DUBm. Gel stained with Coomassie. See also Figure S2.

Figure 2. Structure of DUBm lacking the Sgf11-ZnF shows domain swapping

(A) Structure of DUBm-ΔSgf11-ZnF/Ubp8-S144N shows domain swapping between two DUBm complexes. The two Ubp8 subunits in the complex are colored green and purple, respectively. Other subunits colored as indicated: Sus1 (blue), Sgf11 (orange) Sgf73 (salmon). The disordered Sgf11 linker is represented by a dashed orange line. (B) Initial fit of two DUBm complexes to the electron density map before detection of domain swapping (2Fo-Fc contoured at 1σ, grey, Fo-Fc contoured at 3σ red). (C) Fit of domain-swapped complexes to 2Fo-Fc electron density map. (D) Conformation of Ubp8 in wild-type, intact DUBm (gray) and in domain-swapped complex (green). Residues 133–145 in loop L2 undergo a major conformational change that accompanies domain swapping. (E) Helix in Ubp8 that is buried by the Sgf11-ZnF in the intact DUBm mediates dimerization contacts in the domain-swapped DUBm-ΔSgf11-ZnF.

Figure 3. Ubp8 loops L1 and L2 in the domain-swapped structures

(A) Detailed view of Ubp8 crossover loops (L2) and loop L1 in the domain-swapped DUBmΔSgf11-ZnF-Ubp8^WT structure. Respective Ubp8 monomers are colored purple and green. (B) View as in (A) of the DUBm-ΔSgf11-ZnF-Ubp8^S144N complex. See also Tables S1 and S2.

Figure 4. Effect of domain swapping on the oxyanion hole residue, N141

(A) Superposition of one Ubp8 monomer in the domain-swapped DUBm complex lacking the Sgf11-ZnF (green) with Ubp8 in intact DUBm complex (gray) showing altered position of N141. (B) Superposition as in (A) showing the second Ubp8 monomer (purple) in the domain-swapped complex in which N141 disordered. (C, D) Superpositions as in (A) showing Ubp8 in the domain-swapped DUBm-ΔSgf11-ZnF complex containing Ubp8^S144N. Coloring as in (A)

Figure 5. Assays of wild-type and mutant DUBm oligomerization by velocity sedimentation

(A) Normalized g(s*) analysis for sedimentation of intact wild-type DUBm. Data are fit to a single Gaussian. Residuals shown below. (B) Normalized g(s*) analysis for DUBm-ΔSgf11-ZnF-Ubp8^WT. Black dots indicate raw data, which were fit to a sum of two Gaussians (gray) corresponding to sedimentation of monomeric (red) and dimeric (blue) DUBm. (C) Analysis as in (B) of DUBm-ΔSgf11-ZnF-Ubp8^S144N (D) Analysis as in (B) of DUBm-ΔSgf11-ZnF-Ubp8^S149N (E) Analysis as in (B) of DUBm-ΔSgf11-ZnF-Ubp8^S144N/S149N (F) Superposition of fitted curved from panels A–F; color key in panel inset. See also Figure S3.

Figure 6. Ubp8-N141 is important for DUBm catalysis

(A) Superpositions showing hydrogen bonding of conserved oxyanion hole residue (N141 in Ubp8) to backbone amides. Apo-Ubp8 (2MHH, gray), hUSP8 (2GFO, pink), and hUSP14 (2AYN, gray). (B) Effect of Ubp8 active site mutations on DUBm activity Gel shows time course for cleavage of K48-linked diubiquitin for the intact DUBm-WT, DUBm-Ubp8^C146A, DUBm-Ubp8^N141A, DUBm-ΔSgf11-ZnF-Ubp8^S144N/S149N, and DUBm-ΔSgf11-ZnF-Ubp8^{S144N/S149N/N141A}.

Figure 7. Ubp8 lacks conserved structural elements that could stabilize loop L2

(A) View of intact DUBm with transparent surface depicted for all Ubp8 (green) USP domain residues except loop L2 in Ubp8 (green). Interactions with the Sgf11 (orange) and Sgf73 (pink) zinc fingers help to maintain the observed conformation of loop L2. (B) View of USP14 (2AYN, blue) with transparent surface shown for all residues C-terminal to loop L2. Additional residues in the USP domain bury part of loop L2 and form stabilizing beta sheet interactions. (C) View of hUSP8 (2GFO, magenta) with transparent surface shown for all residues excluding loop L2.