Structural insights into the assembly and function of the SAGA deubiquitinating module

Abstract

SAGA is a transcriptional coactivator complex that is conserved across eukaryotes and performs multiple functions during transcriptional activation and elongation. One role is deubiquitination of histone H2B, and this activity resides in a distinct subcomplex called the deubiquitinating module (DUBm), which contains the ubiquitin-specific protease Ubp8, bound to Sgf11, Sus1, and Sgf73. The deubiquitinating activity depends on the presence of all four DUBm proteins. We report here the 1.90 angstrom resolution crystal structure of the DUBm bound to ubiquitin aldehyde, as well as the 2.45 angstrom resolution structure of the uncomplexed DUBm. The structure reveals an arrangement of protein domains that gives rise to a highly interconnected complex, which is stabilized by eight structural zinc atoms that are critical for enzymatic activity. The structure suggests a model for how interactions with the other DUBm proteins activate Ubp8 and allows us to speculate about how the DUBm binds to monoubiquitinated histone H2B in nucleosomes.

Figure 1. Overall structure of the SAGA DUB module

(A) View of DUB module bound to ubiquitin aldehyde (Ubal). The ZnF-UBP lobe and Usp lobe are labeled. Coloring scheme is Ubp8, green, Sgf11, magenta, Sus1, blue, Sgf73, salmon, Ubal, yellow, with zinc atoms shown as red spheres. Disordered loop residues are indicated with dotted lines. Residues in Sgf73 that are ordered in the structure of the apo DUBm are shown in gray. (B) View of DUB module rotated 180° about the vertical as compared to panel B. (C) View of the ZnF-UBP lobe. The Ubp8 ZnF-UBP domain and Sus1 bind on opposite faces of the Sgf11 N-terminal helix. The Sgf73 residues shown in gray are disordered in the Ubal-bound complex but are ordered in the apo complex. The insertion point for polyglutamine expansion in the human homolog, ATXN7, is indicated by an inverted triangle containing the letter, Q. (D) View of the Usp lobe. The bound ubiquitin is shown in yellow.

Figure 2. Dissociation of the Sgf73 fragment from the DUBm complex

Velocity sedimentation of the DUBm in the presence (blue) and absence (black) of 2 mM EDTA, superimposed with the sedimentation of Sgf73 (-106) alone.

Figure 3. Structural details of the DUB module

(A) Structural zinc coordination in the Ubp8 Usp domain located between α9 and α10. (B) Structural zinc coordination in the Ubp8 Usp domain located between α12 and α13. (C) Superposition of the structure of Sus1/Sgf11^1-33 (gold) (PDB ID 3KIK) with the corresponding residues of Sus1 (blue) and Sgf11 (magenta/pink) in the DUBm. The arrow indicates the additional helical residues (pink) in Sgf11 seen in the DUBm structure.

Figure 4. Interaction between the DUB module and substrate

(A) Alignment of apo Ubp8 (red), Ubp8 (green)-Ubal (yellow), and USP8 (orange). In the apo USP8 structure, the fingers region is collapsed and cannot accommodate ubiquitin without a conformational change. (B) Structural changes in the region of the Ubp8 active site. Residues 228-233 of the Ubp8 apoenzyme (red) are disordered in the absence of ubiquitin and the loop, 421-426, shifts into the groove where the C-terminal tail of ubiquitin (yellow) would bind. The loop, which contains the active site cysteine, C146, maintains the same conformation in the presence and absence of ubiquitin and is stabilized by contacts with the Sgf11 zinc finger (magenta). The other active site residues, H427, N443, and D444, also remain in the same orientation in both structures. (C) Structure of the DUBm showing a surface representation of Sgf73. The color scheme used is the same is in Figure 1. (D) Electrostatic surface potential of the ubiquitin-binding face of the DUBm. The region of strong electropositive surface potential (blue) is due to the Sgf11 zinc finger.