The DNA fragment encoding the WH domain (residue458-577) of ORC2 was obtained by PCR from Homo sapiens brains genomic DNA and cloned into the vector pET28A. The recombinant vector was then transformed into Escherichia coli strain BL21 (DE3). For uniformly 13C-, 15N-labeled recombinant WH domain, cells were cultured at 37°C in LR minimal medium (containing 2.5 g/L 13C-glucose and 0.5 g/L 15NH4Cl as the sole carbon and nitrogen source, respectively) to an OD600 of 1.0, and induced with 1 mM IPTG. They were then incubated at 16℃ for 24 hours. The induced cells were harvested by centrifugation at 5,000 g for 10 min, and resuspended in 35 mL Tris–NaCl buffer (10 mM Tris, 1 M NaCl, 1 mM DTT, pH 7.5). Ecoli were lysed by high pressure and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant after centrifugation was purified with Ni-NTA resin. The target protein was cut off by pTEV. After the tag is cut off, flow the solution through the Ni-NTA resin filled column (QIAGEN) again, separate His-tag from target protein.
The flow through was further purified by superdex 75 (GE Healthcare) in a buffer containing 10 mM Tris, pH 7.5, 1 mM DTT and 150 mM NaCl. Then, a single target protein component was collected through the UV absorption peak, and it was concentrated to a concentration of ~ 20 mg/ mL to collect the NMR signal, and the excess protein can be frozen at -80°C. The final NMR buffer was 20 mM phosphate (pH 7.0), 50 mM NaCl in 10:90% D2O: H2O.
All NMR experiments were recorded at 298K on a Bruker AvanceIII 600 spectrometer equipped with a cryoprobe. The following spectra were recorded to obtain backbone and side chain resonance assignments: 1H-15N HSQC, 1H-13C HSQC, CBCA(CO)NH, CBCANH, HNCA, HN(CO)CA, HNCO, HN(CA)CO, H(CCO)NH-TOCSY, HBHA(CO)NH-TOCSY, (H)C(CO)NH-TOCSY, HCCH-COSY and HCCH-TOCSY. NMR data processing was achieved using NMRpipe and NMRDraw software (Delaglio et al. 1995), and then analyzed with Sparky3 (Goddard et al. 1993).
15N relaxation measurements were also performed on the Bruker AvanceIII 600 spectrometer using a 15N-labeled WH domain of ORC2 sample with a concentration of 0.6 mM at 298 K. The 1H-15N heteronuclear NOE experiments was recorded in an interleaved fashion, alternately with and without proton presaturation in the recovery delay. 15N 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, data deposition and Dynamic Properties of the WH domain
Residues of WH domain were almost identified: in the backbone, 97% of NH and 15N, 100% of 13C, and 90% of 1H chemical shifts were assigned; in the side chain, 96% of the aliphatic and 95% of the aromatic 1H-13C nuclei were assigned. The 1H-15N HSQC spectrum for WH domain was shown in Fig. 1.
The residues in the conserved motif WH of ORC2 were well assigned. The data sets from spectra CBCANH, CBCA(CO)NH, (H)C(CO)NH-TOCSY, HCCH-COSY and HCCH-TOCSY contained the 13C signals for the proline residues. The four proline residues were with normal chemical shifts. The consensus chemical shift index derived from 1Hα, 13Cα, 13Cβ, 13C’ and 15N chemical shifts (Wishart, et al.1994) was shown in Fig. 3. The bands below are the analytical structure of the crystal. This data indicated the presence of four β-sheets and six α-helixes. The chemical shift data have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession number 27643.
The dynamic characteristics of the WH domain in solution were studied by measuring the relaxation time of the WH domain in the free state at T1, T2 and the steady state 1H-15N NOE. The average values of T1 (Fig. 2A), T2 (Fig. 2B), and 1 H-15N NOE for positive values (Fig. 2C) of WH were 0.68 ± 0.02 s, 0.09 ± 0.002 s, and 0.74, respectively. The average 1H-15N NOE value is 0.74, indicating that most of the regions of WH are relatively rigid, corresponding well with the narrow distribution of conformers in the calculated ensemble. Together, the experimental results show that the WH domain in solution is a structural protein with regular skeleton folding. The overall rotational correlation time (τc) of the ~ 15.2KD WH domain is 7.5 ns (as estimated from the average values of T1/T2), suggesting that WH domain is mainly monomeric in solution.