1H, 15N, 13C resonance assignments for proteasome shuttle factor hHR23a

hHR23a (human homolog of Rad23 a) functions in nucleotide excision repair and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like (UBL) domain, an xeroderma pigmentosum C (XPC)-binding domain, and a ubiquitin-associated (UBA) domain preceding and following the XPC-binding domain. Each of the four structural domains are connected by flexible linker regions. We report in this NMR study, the 1H, 15N and 13C resonance assignments for the backbone and sidechain atoms of the hHR23a full-length protein with BioMagResBank accession number 52059. Assignments are 97% and 87% for the backbone (NH, N, C’, Cα, and Hα) and sidechain atoms of the hHR23a structured regions. The secondary structural elements predicted from the NMR data fit well to the hHR23a NMR structure. The assignments described in this manuscript can be used to apply NMR for studies of hHR23a with its binding partners.


Biological context
hHR23a (human homolog of Rad23 a) is one of two human orthologs of yeast radiation sensitivity abnormal 23 (Rad23) that binds to xeroderma pigmentosum C (XPC), forming a complex that functions in nucleotide excision repair of bulky lesions in DNA, such as induced by ultraviolet light exposure (Dantuma et al., 2009).It also acts as a shuttle factor in delivering ubiquitinated proteins to the proteasome (Chen et al., 2021; Osei-Amponsa and Walters, 2022; Wang et al., 2003) and in the nucleus, the two UBA domains of hHR23b have been shown to drive the formation of proteasome-containing biomolecular condensates (Yasuda et al., 2020).hHR23a comprises four well de ned functional domains connected by exible linker regions: an N-terminal ubiquitin-like (UBL) domain followed by an internal ubiquitin-associated (UBA) domain (UBA1), an XPC-binding domain, and a C-terminal UBA domain (UBA2) (Walters et al., 2003).As a ubiquitin-binding shuttle factor, hHR23a/Rad23 recognizes ubiquitinated substrates with its two UBA domains (Bertolaet et al., 2001;Wang et al., 2003) and the proteasome subunits Rpn1, Rpn10 and Rpn13 with its UBL domain (Elsasser et al., 2002;Husnjak et al., 2008;Rosenzweig et al., 2012;Gomez et al., 2011;Mueller and Feigon, 2003;Shi et al., 2016;Chen et al., 2016).The hHR23a UBL domain interacts dynamically with its two UBA domains and this interaction is broken when hHR23a binds to either proteasome component Rpn10 or ubiquitin (Walters et al., 2003;Wang et al., 2003).hHR23a can also bind another shuttle factor UBQLN2 and form heterodimer by UBL/UBA domain interactions (Kang et al., 2007b).The structure of hHR23a has been extensively studied (Dieckmann et al., 1998 (Jiang et al., 2017), and a construct that includes the XPC-binding and UBA2 domains (BMRB 27978) (Byeon et al., 2020).Here, we report the chemical shift assignments for full-length hHR23a, which is related to our previous reported full-length hHR23a NMR structure (Walters et al., 2003).These assignments may serve as a foundation for NMR studies of hHR23a interactions with proteasome subunits Rpn1, Rpn10, and Rpn13 or other ubiquitin receptors, such as Ddi1 or UBQLN2.

Expression and puri cation of hHR23a
Full-length hHR23a was subcloned between BamHI and NotI restriction sites of the pGEX-6P-1 vector (Cytiva 27-1542-01) in frame with an N-terminal glutathione S-transferase (GST) and a PreScission protease cleavage site.The plasmid was transformed into E. coli strain BL21(DE3) (Thermo Fisher Scienti c C600003) with selection by ampicillin.The transformed colonies were grown in 10 mL of Luria-Bertani Broth (LB) medium (ampicillin 100 µg/ml) overnight at 37°C with shaking and centrifuged for 10 minutes at 2,000 g.The bacterial pellets were then gently resuspended and diluted at 1:100 ratio with 1 L of M9 minimal media supplemented with 1 g/L 15 N ammonium chloride (Sigma-Aldrich) and 3 g/L 13 C glucose (Sigma-Aldrich) as the sole nitrogen and carbon sources, or 1 g/L unlabeled ammonium chloride and 3 g/L 13 C glucose, or 1 g/L 15 N ammonium chloride and 3 g/L 13 C glucose in 50%2 H 2 O (Sigma-Aldrich) / 50%1 H 2 O, as described in (Chen and Walters, 2012).Cells were grown at 37°C with shaking until they reached an OD 600 of 0.5-0.6 at which point isopropyl β-D-1-thiogalactopyranoside (UBPBio) was added to a nal concentration of 0.4 mM to induce protein expression at 17°C overnight.The bacteria were harvested by spinning down for 30 minutes at 5,000 g and 4°C in a Beckman Coulter J6-M1 centrifuge with a JS-4.2 rotor.The harvested cell pellets were frozen in liquid nitrogen and stored at -80°C until puri cation.
Cell pellets were resuspended in lysis buffer (20 mM NaPO 4 at pH 6.5, 300 mM NaCl, 2 mM DTT) supplemented with protease inhibitor cocktail tablets (Roche Diagnostics 11836153001).Resuspended cells were sonicated and centrifuged for 30 min at 27,000 g and 4°C.The supernatant was incubated with pre-washed glutathione-Sepharose beads (Cytiva 17075605) for 3 hours at 4°C with agitation.The beads were washed extensively in lysis buffer and incubated overnight with PreScission protease (Cytiva 27084301) at 4°C with agitation to release hHR23a protein from the GST tag.hHR23a proteins were eluted from the beads in lysis buffer and further puri cation was achieved by size exclusion chromatography on an FPLC ÄKTA pure system (Cytiva) equipped with a HiLoad 16/600 Superdex 200 prep grade column (Cytiva) in FPLC buffer (20 mM NaPO 4 at pH 6.5, 100 mM NaCl, 2 mM DTT).hHR23a proteins were concentrated by Amicon Ultra-15 lters with a 3 kDa cutoff (EMD Millipore UFC900324) to ~ 0.5 mM.
All NMR data processing was performed with NMRpipe (Delaglio et al., 1995), and spectra were visualized and analyzed with XEASY (Bartels et al., 1995).Secondary structure was assessed by comparing chemical shift values of C α and C' atoms to random coil positions to generate a chemical shift index (CSI) (Wishart and Sykes, 1994).

of assignments and data deposition
Figure 1 shows an assigned 2D 1 H-15 N HSQC spectrum of 15 N, 13 C, 50% 2 H-hHR23a at pH 6.5 and 25°C.
The secondary structure of the hHR23a was predicted by plotting the difference between the chemical shift values of carbonyl and Cα atoms relative to those of randomly coiled values (Wishart and Sykes, 1994) (Fig. 2).Five β strands and eleven α helices were predicted, consistent with the NMR structure of hHR23a (PDB 1OQY).All secondary structure was further con rmed by NOE interactions from 15 N-edited NOESY-HSQC and 13 C-edited NOESY-HSQC spectra.

Figure 1
Figure 1 hHR23a domain layout and 2D 1 H-15 N HSQC spectrum (A) Domain organization of hHR23a with the UBL, UBA1, XPC-binding, and UBA2 domain shaded in blue, orange, green, and red, respectively.(B) 2D 1 H-15 N HSQC spectrum of 15 N, 13 C, 50% 2 H-labeled hHR23a (0.5 mM) in 20 mM NaPO 4 at pH 6.5, 100 mM NaCl, 2 mM DTT, 0.1% NaN 3 and 10% 2 H 2 O / 90% 1 H 2 O collected at 25°C on a Varian INOVA 800 MHz spectrometer.Residue type and sequence position are included for the signals corresponding to backbone amides and labeled in blue, orange, green, red, and grey for UBL, UBA1, XPC-binding, UBA2 domain, and linker region, respectively.A dashed box indicates the enlarged region shown in the panel on the right.

Figure 2 1 hHR23a
Figure 2 Secondary structure of the hHR23a obtained from the NMR structure 1OQY (top panel) or as predicted by ΔδC' (middle panel) and ΔδCα (bottom panel) are displayed along the amino acid sequence.ΔδC' and ΔδCα are calculated by subtracting randomly coiled values for the corresponding amino acid

Figure 2 Secondary
Figure 2