We previously described expression and purification of nsp3 CoV-Y (residues 1638–1945) (Altincekic et al. 2021). This construct provided good quality 1H-15N TROSY-HSQC spectra but a long N-terminal unstructured region (approximately 30 residues) complicated the assignment. In this work we used a shorter construct of nsp3 CoV-Y that corresponds to residues 1660–1945 based on the NCBI reference sequence YP_009742610.1. The gene sequence was E. coli codon optimized and cloned into a pET28b( +) vector containing a removable tobacco etch virus (TEV) protease recognition site. After TEV cleavage, the final construct contained an artificial N-terminal glycine residue preceding the native CoV-Y sequence.
For backbone assignment we produced partially deuterated U-[15N-13C]- nsp3 CoV-Y protein by growing BL21 (DE3) E. coli cells transformed with the pET28b( +) plasmid encoding CoV-Y domain in M9 media using D2O with 1 mg/L U-[15N]-labeled ammonium chloride and 4 mg/L U-[13C]-labeled D-glucose as sole sources of nitrogen and carbon, respectively.
For methyl labeling of the nsp3 CoV-Y domain we used the procedure described by Tugarinov et al. (Tugarinov and Kay 2004; Tugarinov et al. 2005). A U-[15N,13C,2H], Ile δ1-[13CH3], Leu, Val-[13CH3,12CD3]-labeled sample of nsp3 CoV-Y (U-[15N,13C,2H]-ILV) was produced using D2O M9 media with 1 mg/L U-[15N]-labeled ammonium chloride and 4 mg/L U-[13C,2H]-labeled D-glucose. For selective methyl protonation 70 mg/L of [13C4; 3,3-2H2]-alpha-ketobuterate and 140 mg/L of [1,2,3,4-13C4; 3,4′,4′,4′-2H4]-alpha-ketoisovaleriate were added 1 hour prior to induction. All isotopes were purchased from Cambridge Isotope Laboratories, Inc.
The protein was purified as described previously (Altincekic et al. 2021) with two modifications: (I) we added 1 mM of tris (2-carboxyethyl) phosphine (TCEP) after TEV-cleavage of the His6-tag to reduce cysteine side chains; (II) we changed the final buffer because the computed pI was reduced from 7.1 to 6.6 for the new shorter construct. All NMR samples were prepared in 20 mM MOPS buffer pH 6.4, 100 mM LiBr, 2 mM DTT and 0.01% NaN3.
All NMR experiments were recorded at 25 °C on a Varian Inova 800 MHz spectrometer equipped with a cryogenic triple-resonance probe. For the backbone resonance assignments a sample of 0.35 mM partially deuterated U-[15N-13C]- nsp3 CoV-Y in NMR buffer with 10% D2O was used. A set of NMR experiments for backbone 15N, 13C and 1H resonance assignment consisted of 2D 1H-15N TROSY-HSQC and three-dimensional non-uniformly sampled TROSY-based HNCO (15%), HNCACO (15%), HNCA (16%), HNCACB (14%) (Yang and Kay 1999) and 15N-edited NOESY-HSQC (20%) with 140 ms mixing time (Zhang et al. 1994). For the assignment of Ile δ1, Leu δ1,2 and Val γ1,2 methyl groups a 0.25 mM U-[15N,13C,2H]-ILV sample was used. We recorded 2D 1H-13C HSQC and 3D HMCMCGCBCA (Tugarinov and Kay 2003) acquired with 20% NUS sampling coverage.
All spectra were processed using NMRpipe (Delaglio et al. 1995) and SMILE for NUS spectral reconstruction (Ying et al. 2017).
Assignment and data deposition
Analysis of the spectra and backbone resonance assignment were performed manually using CARA (Keller 2004). The 1H-15N TROSY-HSQC spectrum of nsp3 CoV-Y domain shows excellent signal dispersion for a 286-residue protein (Fig. 1). Backbone assignment is nearly complete (97% HN, 94% N, 98% C′, 98% Cα, 90% Cβ). There are nine missing residue assignments, K1660, N1680, N1854, T1858, Y1859, N1934-T1937 in the 1H-15N TROSY-HSQC spectrum. The HN peaks for missing residues are probably broadened beyond detection in the 1H-15N TROSY-HSQC. A few unassigned low intensity peaks in 1H-15N TROSY-HSQC spectrum don’t provide identifiable cross-peaks in 3D spectra for reliable correlation.
Measured backbone chemical shifts were used as input for Talos + (Shen and Bax 2015) to calculate the random-coil-index-derived order parameters (RCI-S2) and secondary structure propensities (Fig. 2). Our results suggest that the CoV-Y domain possesses a well-ordered globular α/β fold with flexible termini (eight N-terminal residues and four C-terminal residues). These terminal residues as well as residues 1932–1936 have RCI-S2 < 0.65 (Fig. 2). This probably explains why peaks corresponding to N1934-T1937 are broadened beyond detection in the 1H-15N TROSY-HSQC spectrum.
Methyl groups assignment and figure preparation were done with CCPN Analysis (Vranken et al. 2005). All 14 Ile δ1 and 25 Leu δ1,2 groups were assigned as well as 24 out of 28 Val γ1,2 (Fig. 3). Due to lack of backbone assignment for V1935 and V1936, partial assignment of V1933, and overlap in spectra, the methyl groups of V1777, V1933, V1935 and V1936 were not assigned.
All obtained backbone and methyl chemical shifts of SARS-CoV-2 Nsp3 CoV-Y have been deposited at the BMRB under accession number 51074.
Our results provide a foundation for future NMR studies of nsp3 CoV-Y domain to identify interaction partners and to understand the molecular mechanism of DMV formation.