Transient interdomain interactions in free USP14 shape its conformational ensemble

Abstract

The deubiquitinase (DUB) ubiquitin‐specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin‐like (Ubl) domain separated by a 25‐residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the proteasome, where the Ubl and USP domains of USP14 bind to the Rpn1 and Rpt1/Rpt2 units, respectively. No structure of full‐length USP14 in the absence of proteasome has yet been presented, however, earlier work has described how transient interactions between Ubl and USP domains in USP4 and USP7 regulate DUB activity. To better understand the roles of the Ubl and USP domains in USP14, we studied the Ubl domain alone and in full‐length USP14 by nuclear magnetic resonance spectroscopy and used small angle x‐ray scattering and molecular modeling to visualize the entire USP14 protein ensemble. Jointly, our results show how transient interdomain interactions between the Ubl and USP domains of USP14 predispose its conformational ensemble for proteasome binding, which may have functional implications for proteasome regulation and may be exploited in the design of future USP14 inhibitors.

Dynamic regions allosterically connect the USP14 active site with the proteasome interaction surface

“…Overall, the solution secondary structure compares well to that presented in the crystal structures of the USP14USP in the absence and presence of Ub (PDB-ID 2AYN, 2AYO [12]) (Figure 2A). An even better match was observed with the AlphaFold model (Figure 2A), in agreement with its excellent fit to SAXS solution data [39].…”

“…Although this USP14 region has not previously been assigned functional relevance, the helix corresponding to helix α4 in USP12 adopts shifted positions in different crystals structures, suggesting dynamics [54]. We note that this region corresponds to one of the dynamic USP touchpoints of the USP14 Ubl in full-length USP14 as determined through joint probing by NMR and SAXS techniques[39]. The possible functional relevance of this intersegmental connection will need to be further investigated.…”

“…This model contains all USP14 USP residues, including loops (BL1-3, SL and PKL) indicated to be of importance for function [11,12,18] (Figure 1C) and previously unresolved regions in crystal[12] or cryo-EM structures [11]. We recently validated this USP14 USP model as the best fit to small-angle X-ray scattering (SAXS) data in solution [39]. Most of the unassigned residues are in surface loops or are deeply buried in the β-sheet core (Figure 1D) .…”

“…Complementary assignments for loops were achieved by combining the new experiments with spectral information from the 13 C, 15 N labeled USP14 1-494 , where crowding was less of an issue. [39] CheSPI [40] was utilized for secondary structure populations based on N, H N , C α , C β , and C’ chemical shifts.…”

“…TROSY-optimized 15 N R 1 , R 2 and hetNOE experiments [42] were collected as pseudo-3D at 25°C using a Bruker Avance III HD 900 MHz spectrometer equipped with 3 mm TCI cryoprobe at the Swedish NMR centre, on USP14 99-494 with a concentration of 0.89 mM in same buffer as above. For the relaxation measurements, the USP construct was slightly shortened in the N-terminus, compared to the assignment experiments, to exclude the flexible region 91-98 [39]. The R 1 and experiment were collected using 12 different relaxation delays and 2 duplicates: 0.08, 0.08, 0.16, 0.24, 0.32, 0.4, 0.4, 0.56, 0.72, 0.8, 1.04, 1.2, 1.6 and 2.4 s and the R 2 experiment with 10 different relaxation rates, one duplicate and one triplicate: 0.008, 0.016, 0.016, 0.016, 0.031, 0.047, 0.063, 0.094, 0.110, 0.110, 0.125, 0.156, 0.203 and 0.250 s for R 2 .…”