1H, 15N, 13C backbone and Cβ resonance assignments for UBQLN1 UBA and UBAA domains

UBQLN1 functions in autophagy and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like domain (UBL), a C-terminal ubiquitin-associated domain (UBA), and a flexible central region which functions as a chaperone to prevent protein aggregation. Here, we report the 1H, 15N, and 13C resonance assignments for the backbone (NH, N, C’, Cα, and Hα) and sidechain Cβ atoms of the UBQLN1 UBA and an N-terminally adjacent segment called the UBA-adjacent domain (UBAA). We find a subset of the resonances corresponding to the UBAA to have concentration-dependent chemical shifts, likely due to self-association. We also find the backbone amide nitrogen of T572 to be shifted upfield relative to the average value for a threonine amide nitrogen, a phenomenon likely caused by T572 Hγ1 engagement in a hydrogen bond with adjacent backbone carbonyl atoms. The assignments described in this manuscript can be used to study the protein dynamics of the UBQLN1 UBA and UBAA as well as the interaction of these domains with other proteins.

Sidechain NH 2 groups of asparagine and glutamine are also labeled 1 H, 15 N, 13 C backbone and Cβ resonance assignments for UBQLN1 UBA and UBAA domains

Methods and experiments
Expression and purification  and F547Y) were synthesized with codon optimization for expression in E. coli and inserted into the pGEX-6P-3 vector between the EcoRI and XhoI sites in frame with the PreScission protease cleavage site (GenScript). Plasmids were transformed into E. coli strain BL21(DE3) (Invitrogen C600003) in the presence of 100 µg/mL ampicillin. Transformants were grown in 10 mL of Luria-Bertani Broth (LB) overnight at 37 °C with shaking and centrifuged for 10 min at 2000 g. Bacterial pellets containing UBQLN1 constructs were gently resuspended into 1 L of M9 minimal media containing 1 g/L 15 N ammonium chloride (Cambridge Isotope Laboratories NLM-467-1) as their only nitrogen source. Media used for UBQLN1 (514-586) additionally contained 2 g/L 13 C glucose (Cambridge Isotope Laboratories CLM-1396) as the sole carbon source and was grown in 80% 2 H 2 O/20% 1 H 2 O ( 2 H 2 O is from Cambridge Isotope Laboratories DLM-4-99-1000). Cells were grown at 37 °C with shaking until they reached an OD 600 of 0.5-0.6 at which point IPTG was added to a concentration of 0.4 mM to induce protein expression at 17 °C overnight. Bacteria were pelleted at 4,000 rpm for 40 min at 4 °C in a Beckman Coulter J6-M1 centrifuge fitted with a JS-4.2 rotor and frozen at -80 °C until purification. Cell pellets were resuspended in lysis buffer (20 mM at Tris pH 7.6, 300 mM NaCl, 5 mM DTT, and Roche Complete Mini protease inhibitor cocktail (Roche Diagnostics 11836153001)). Resuspended bacteria were sonicated and centrifuged at 15,000 rpm (~ 27,216 g) at 4 °C for 30 min. Supernatants were incubated with glutathione Sepharose beads (Cytiva 17-0756-05) with fresh protease cocktail inhibitor added, for 3 h at 4 °C with agitation. Beads were washed 4-5 times in lysis buffer without protease inhibitors and incubated overnight with PreScission protease (Cytiva 27084301) at 4 °C with agitation to release UBQLN protein from the GST tag. UBQLN1 proteins were eluted from the beads in lysis buffer and then purified further by size exclusion chromatography on an ÄKTA pure FPLC system (Cytiva) using a HiLoad 16/600 Superdex75 prep grade column in FPLC buffer (10 mM MOPS at pH 6.5, 50 mM NaCl, 5 mM DTT, and 10 µM zinc sulfate). UBQLN1 proteins were concentrated with Amicon Ultra-15 filters with a 3 kDa cutoff (EMD Millipore UFC900324). Liquid chromatography-mass spectrometry (LC-MS) was performed with protein samples (10 µM) with 10% acetonitrile on a 6100 Series Quadrupole LC-MS (Agilent Technologies, Inc.), equipped with an electrospray source and operated in the positive ion mode. Data acquisition and analyses were performed using OpenLAB CDS ChemStation Edition C.01.05 (Agilent Technologies, Inc.). Based on the Mass (9370.20 Da) from LC-MS (Fig. 2), deuterium labeling of UBQLN1 (514-586) was estimated to be ~ 64%.

Extent of assignments and data deposition
Assignments were completed for all backbone atoms of UBQLN1 514-586. Excluding residual non-native residues left behind from removal of the GST tag, 100% of backbone N, HN, C', Cα, and Cβ atoms were assigned as well as 96% of Hα atoms (missing T514, H522, and P543). The 15 N-dispersed NOESY experiment was used to validate Hα assignments. The F547Y substitution was introduced into the UBQLN1 514-589 construct to enable protein quantitation at 280 nm absorbance, and assignment of UBQLN1 514-589, F547Y was guided by assignments made for UBQLN1 514-586 (Fig. 1B). Of note, the nitrogen of the T572 backbone amide displays a chemical shift value of 105.2 ppm, which is ~1.6 standard deviations upfield compared to the average value for a threonine amide nitrogen (115.4 p.p.m., according to the BMRB website, calculated from the BMRB database, https://bmrb.io/ref_info/csstats). In concurrent work (Buel et al. 2023), we find that Hγ1 of T572 undergoes hydrogen bonding, likely to surrounding backbone carbonyls, and we expect that the upfield shifting of the T572 amide nitrogen is reflective of higher electron shielding afforded by the T572 Hγ1 hydrogen bond.
While working with UBQLN1 514-589, F547Y, residues in the region of H522-N537 were found to have concentration-dependent chemical shifts (Fig. 3A,B). We think this is due to self-association of UBQLN1 514-589, F547Y monomers in solution. We found in concurrent studies that protein binding to the nearby UBA allosterically induces changes in the UBAA region as observed through chemical shift perturbation analyses (Buel et al. 2023), and we therefore recommend exercising caution when interpreting chemical shift perturbation data in this region. Assignments for UBQLN1 514-586 have been deposited in the Biological Magnetic Resonance Data Bank (BMRB) under accession code 51768. Assignments for UBQLN1 514-589, F547Y were deposited to the BMRB at two concentrations: 50 µM (BMRB 51769) and 600 µM (BMRB 51770).

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Competing interests
The authors declare that they have no conflict of interest.
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