Structural characterization of the interaction of Ubp6 with the 26S proteasome

Abstract

In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.

Keywords: conformational switching; proteolysis; proteostasis; quality control.

Fig. 1.

26S proteasome reconstructions for different buffers. (A) Reconstructions from 26S proteasomes alone, filtered to 15-Å resolution (6). (B–D) Reconstructions from 26S–Ubp6 (B), 26S–Ubp6–UbAld (C), and 26S–UbAld (D) datasets obtained in the present study, all filtered to ∼15-Å resolution. The black arrow in B indicates the blurred PCI horseshoe. The dotted orange ellipse and the orange arrow in C mark the extra density seen for the 26S–Ubp6–UbAld reconstruction.

Fig. 2.

Classification of datasets into s1 and s2 states. The bars indicate the relative frequencies of s1 and s2 states. (A) The s1 and s2 reconstructions obtained from 26S proteasomes alone (13). The different subunits of the regulatory particles are indicated for the s1 state. (B–D) As in A, for the 26S–Ubp6 (B), 26S–Ubp6–UbAld (C), and 26S–UbAld (D) datasets. The dotted orange ellipses and the orange arrows mark the extra density seen for the 26S–Ubp6–UbAld reconstruction shown in Fig. 1C.

Fig. 3.

Density of proteasome-bound Ubp6–UbAld. (A) Refined density of the 26S–Ubp6–UbAld complex from 26S–Ubp6–UbAld dataset. (B) Comparison of the atomic model of the s2 state and the density. The difference between the two is rendered as an isosurface. The orange density is specific for the 26S–Ubp6–UbAld data, and the gray area corresponds to the disordered N termini of the coiled-coils of Rpt4/5, which also are present in the EM density from 26S proteasomes alone but are not included in the model.

Fig. 4.

Fit of the Ubp6 USP domain in the refined 26S–Ubp6–UbAld density. (A) Assessment of orientation specificity of fitting. The Z-scores of the correlation are plotted against the angular distance to the best-fitting result, which is more than six standard deviations above the mean value. The coordinates are colored according to the distance of the cross-linked residues of the USP domain and Rpt1 (Table S1). (B) Visualization of the cross-link for the three best-scoring solutions in A (orange: Ubp6; purple: UbAld; blue: Rpt1; brown: Rpt1; cyan: Rpt5; red: CP).

Fig. 5.

Model of proteasome-bound Ubp6–UbAld. (A and B) Best-fitting model from Fig. 4 positioned in the extra density-containing class from the 26S–Ubp6–UbAld and 26S–Ubp6 datasets, respectively. (C, Upper) Atomic model of the 26S–Ubp6–UbAld complex seen in two different views. (Lower) The boxed region is enlarged for better visualization of Ubp6^104-499 and UbAld. In the enlarged top view (Lower Right) Rpn13, Rpn2, Rpn8, and the PCI horseshoe have been removed for clarity.