Resonance assignments of the PUB domain of the RNF31 protein

E3 ubiquitin protein ligase RNF31 is present in human proteins and is involved in linear ubiquitin chain assembly complex (LUBAC) activity and cell growth. RNF31 is involved in ubiquitination, which is the post-translational modification of proteins. Ubiquitin molecules connect with amino acid residues of target proteins under the action of ubiquitin-activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3, so as to achieve certain physiological functions. The abnormal expression of ubiquitination promotes the formation of cancer. In studies of breast cancer, RNF31 mRNA levels were found to be higher in cancer cells than in other tissues. The PUB domain of RNF31 is the binding site of the ubiquitin thioesterase otulin. Here, we report the backbone and side-chain resonance assignments of the PUB domain of RNF31 and study the backbone relaxation of the domain. These studies will contribute to further understanding of the structural and functional relationship of RNF31 protein, which may also be a target for drug research.

584 of RNF31 changes to histidine (Q584H) and glutamine 622 changes to leucine (Q622L), there is a stronger interaction between RNF31 and RBCK1, resulting in increased E3 ligase activity of the complex (Grumati et al. 2014).Excessive linear ubiquitin chain signaling affects the NF-κB pathway, which is overactivated and is responsible for cancer cell malignancy (Yang et al. 2014;Grumati et al. 2014).
Therefore, it is important to study the structure and function of RNF31 in the formation of LUBAC.A partial fragment of RNF31, the PUB domain (Schaeffer et al. 2014), was studied here.This domain is closely related to the ubiquitination involving RNF31.Inhibiting the formation of the LUBAC complex can slow the growth of cancerous cells (Thompson et al., 2004).The resonance assignment of the PUB domain of RNF31 will help us further understand the structure and the relationship between the structure and function of RNF31.

Methods and experiments
The DNA fragment encoding the PUB domain (residue 4-179) of RNF31 from Homo sapiens was obtained by PCR from Homo sapiens brains genomic DNA using the two primers 5'-GGGAATTCCATATGGAACGCGCGTTTCTG-3' and 5'-CCGCTCGAGTTATTCCAGCAGTTG-3' and

Biological context
Linear ubiquitin chain Assembly Complex (LUBAC) is a type of ubiquitin ligase (Oikawa et al. 2020;Wang et al. 2020).LUBAC-specific generation of N-terminal MET1linked linear ubiquitin chains has a regulatory role in acquired and innate immune responses (Schaeffer et al. 2014;Elliott et al. 2014).RNF31 plays an important role in LUBAC-mediated ubiquitination.The ubiquitin related UBA domain of RNF31 binds to the ubiquitin like UBL domain of RBCK1, and the specific interactions between the two domains are critical for the formation of the LUBAC (Zhu et al. 2014;Kharman-Biz et al. 2018).UBA is a highly conserved region of RNF31, responsible for the interactions between RNF31 and RBCK1.When glutamine cloned into the NdeI/XhoI-cleaved vector pGEX-4T-1.Recombinant vector was introduced into Escherichia coli BL21.The recombinant PUB domain possesses 176 amino acids.To label the PUB domain with 13 C and 15 N, cells were cultured at 37℃ in M9 minimal medium (containing 2.5 g/L 99% 13 C-glucose and 0.5 g/L 99% 15 NH 4 Cl as the sole carbon and nitrogen source, respectively) to an OD 600 of 1.0, and induced with 0.5 mM IPTG for 24 h.The induced cells were harvested by centrifugation at 5,000 g for 10 min, and resuspended in 35 mL Tris-NaCl buffer (20 mM Tris, 1 M NaCl, pH 7.3).Cells were lysed by high pressure and centrifuged at 12,000 rpm for 30 min at 4℃. GSTSep glutathione agarose resin was added to the supernatant and incubated for 24 h.Protein impurity is released at the end of incubation.30mL Tris-NaCl (20 mM Tris, 300 mM NaCl, pH 7.3) suspension was then added to GSTSep glutathione agarose resin, followed by TEV enzyme and 0.2 mM DTT, digested at 4℃ for 20 h.The target protein was washed with 20 mL of buffer (20 mM Tris,300 mM NaCl, pH 7.3) and further purified by gel filtration with superdex 75 column (Amersham).The buffer passing through the gel column contains 200 mM NaCl and 20 mM Na 2 HPO 4 (pH 7.0).The 0.5 mM prepared protein sample was dissolved in final NMR buffer containing 20 mM phosphate (pH 6.0) and 200 mM NaCl in 10:90% D 2 O: H 2 O.
15 N relaxation measurements were also performed on the Bruker AvanceIII 600 spectrometer using 15 N-labeled PUB domain with a concentration of 0.6 mM at 298 K.The 1 H-15 N heteronuclear NOE experiment was recorded in an interleaved fashion, alternately with and without proton presaturation in the recovery delay.The proton saturation time, D1, was set to 5 s. 15 N longitudinal relaxation times (T1) and transverse relaxation times (T2) were derived from eight spectra with different values for the relaxation delay (11, 61, 142, 243, 362, 523, 753, and 1147 ms) and seven relaxation delays (0, 17.6, 35.2, 52.8, 70.4, 105.6, and 140.8 ms), respectively.T1 and T2 values were extracted and fitted using a curve-fitting subroutine included in the program Sparky3 (Goddard et al. 1993).

Extent of assignment and data deposition
Residues of the PUB domain were almost all identified: The identification of the RNF31 PUB domain was approximately 99% complete, with approximately 98% of the backbone atoms and 92% of the side chain atoms identified.The 1 H-15 N HSQC spectrum for the PUB domain is shown in Fig. 1.The residues in the conserved region of the PUB domain are well assigned.The data of (H)C(CO)NH-TOCSY, HCCH-COSY, HCCH-TOCSY and 1 H-13 C HSQC

Dynamic properties of the PUB domain
To study the dynamics of the PUB domain, we measured the 15 N longitudinal (T1) and transverse (T2) relaxation times and the steady-state 1 H-15 N NOE of the PUB domain in the free state.The data for some amino acids were not obtained, due to spectral overlap.However, the overall data are relatively complete, which can still explain the general dynamic changes of the PUB domain.The average values of T1, T2 and 1 H-15 N NOE (Fig. 3) of the PUB domain were 0.96 s, 0.06 s and 0.77, respectively.The steady-state 1 H-15 N NOE is an excellent measure of flexibility in proteins.Together, the experimental results show that the RNF31-PUB protein backbone has a regular secondary structure.

Fig. 3
Fig. 3 Backbone dynamics of 0.6 mM 15 N-labeled RNF31 PUB domain alone at 298 K. T1(A), T2(B) and 1 H-15 N NOE(C) values of the backbone amide resonances of the RNF31 PUB domain are plotted by residue number