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.

Keywords: DUB; NMR; SAXS; molecular modeling; protein dynamics.

FIGURE 1

USP14 working constructs and Ubl/ubiquitin models and structures. (a) Schematic illustration of USP14 domain architecture and the constructs used in this study. (b) AlphaFold model of human USP14‐Ubl (residues 1–80, yellow), and NMR structures of murine USP14‐Ubl (residues 4–86, green, PDB‐ID 1WGG) and human ubiquitin (red, PDB‐ID 1UBQ). USP14, ubiquitin‐specific protease 14.

FIGURE 2

NMR assignment and secondary structure assessment of the USP14 Ubl domain alone and in the context of full‐length USP14. (a) HSQC spectra of USP14_1–80 (yellow) and USP14_1–494 (purple) with resonances assigned to their respective residue. (b) Secondary structure populations of the Ubl domain and linker derived by CheSPI for human USP14_1–80 and USP14_1–494, and their corresponding AlphaFold models colored according to their respective CheSPI plots. NMR, nuclear magnetic resonance; USP14, ubiquitin‐specific protease 14.

FIGURE 3

Comparison of chemical shifts for the Ubl domain in USP14_1–80 and USP14_1–494. (a) Selection of NMR resonances from panel (b) in the USP14‐Ubl domain displaying significant CSPs between USP14_1–80 (yellow) and USP14_1–494 (purple). (b) HSQC spectra of USP14_1–80 superimposed onto USP14_1–494. (c) CSP values comparing the Ubl domain in USP14_1–80 and in the context of USP14_1–494. The solid line represents the trimmed mean, with corresponding standard deviation (2σ) as dashed line (see Section 4 for details). CSPs larger than 2σ from the trimmed mean are considered significant and are shown as black spheres on the USP14‐Ubl AlphaFold model. CSPs, chemical shift perturbations; NMR, nuclear magnetic resonance; USP14, ubiquitin‐specific protease 14.

FIGURE 4

NMR relaxation evaluation of the USP14‐Ubl domain alone and in the context of full‐length USP14. (a) AlphaFold model of USP14‐Ubl (residues 1–75). Ω_49–67‐loop highlighted in copper red. (b) HetNOE values of USP14_1–80 (yellow) and USP14_1–494 (purple). Dashed line indicates the value 0.65 which was used as cutoff for τ _c calculations. Residues below 0.65 for the respective USP14 constructs are shown as spheres. (c and d) R ₁ and R ₂ relaxations rates colored as in (b). Dashed lines represent 2σ from mean using a standard deviation‐based trimming process (see Section 4 for details). Residues above 2σ are indicated as blue (USP14_1–494) or red (USP14_1–80) spheres, and residues below 2σ as purple or yellow. (e) S ² values estimated from TENSOR2. (f) Per‐residue plot of R ₁ R ₂ as a function of R ₂/R ₁, with annotated residues suggested to show motions in slower timescales in one or both contexts, based on elevated values of the R ₁ R ₂ product (Kneller et al., 2002). hetNOE, {¹H}–¹⁵N Overhauser effect; NMR, nuclear magnetic resonance; USP14, ubiquitin‐specific protease 14.

FIGURE 5

USP14‐USP structures and models compared to SAXS data. (a) AlphaFold model of USP14‐USP (residues 91–494, from AF‐P54578‐F1) with loops lacking electron densities in crystal structures highlighted in blue. (b) CRYSOL fits of USP14‐USP models and crystal structures to experimental SAXS data. Bound ligands and waters were removed prior to fit. Chain A was used for structures containing several chains. SAXS, small‐angle x‐ray scattering.

FIGURE 6

SAXS‐based molecular modeling describes the conformational ensemble of full‐length USP14. (a) Starting set of 13,000 generated models of full‐length USP14 superimposed on the USP domain, where spheres represent the center of mass for each Ubl domain. Sphere colors indicate how well each model fits USP14_1–494 SAXS data, with red for χ ² > 5. (b) Projected contour plot of χ ² for all models in the starting set as a function of distance and positioning between the center of mass for the Ubl and USP domains, with coordinate system as in (a) and the USP domain center of mass in origo. (c) Conformational SAXS‐based ensemble of 30 iBME‐selected USP14 models represented as center‐of‐mass spheres as in (a), and superimposed on the USP domain. The sphere volume indicates the model weight in the ensemble and the sphere color how well each individual model fits USP14_1–494 SAXS data, according to the rainbow bar. Models are categorized in three groups depending on the Ubl domain position relative the USP domain and have distinct R _gs (see Figure S4). (d) Weighted fit of the USP14 ensemble to USP14_1–494 SAXS data. (e) The USP14 SAXS‐based ensemble from (c) illustrated in full cartoon, structurally aligned onto the USP domain, and with Ubl domains colored as in (c). (f) USP14 conformational ensemble as in (c), aligned to the USP14‐USP domain in a cryo‐EM structure of the proteasome (PDB‐ID 7W3H). The Ubl interaction site in Rpn1 is indicated by wheat‐colored spheres. iBME, iterative Bayesian/Maximum Entropy; SAXS, small‐angle x‐ray scattering; USP14, ubiquitin‐specific protease 14.