Oligomeric State of β-Coronavirus Non-Structural Protein 10 Stimulators Studied by Small Angle X-ray Scattering

Abstract

The β-coronavirus family, encompassing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS), and Middle East Respiratory Syndrome Coronavirus (MERS), has triggered pandemics within the last two decades. With the possibility of future pandemics, studying the coronavirus family members is necessary to improve knowledge and treatment. These viruses possess 16 non-structural proteins, many of which play crucial roles in viral replication and in other vital functions. One such vital protein is non-structural protein 10 (nsp10), acting as a pivotal stimulator of nsp14 and nsp16, thereby influencing RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to unraveling its multifunctional roles. Our study involves the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. To elucidate their oligomeric state, we employed a combination of Multi-detection Size exclusion chromatography (Multi-detection SEC) with multi-angle static light scattering (MALS) and small angle X-ray scattering (SAXS) techniques. Our findings reveal that full-length nsp10s primarily exist as monomers in solution, while truncated versions tend to oligomerise. SAXS experiments reveal a globular shape for nsp10, a trait conserved in all three coronaviruses, although MERS nsp10, diverges most from SARS and SARS-CoV-2 nsp10s. In summary, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 exhibit a globular and predominantly monomeric state in solution.

Keywords: COVID-19; SARS-CoV-2; SAXS; conformational changes; non-structural proteins; nsp10; oligomeric state.

Figure 1

Protein sequence and structural alignment of full-length nsp10 proteins from SARS-CoV-2, SARS and MERS. Identical residues are shown in white on a red background, similar residues are shaded in red and distinct residues are displayed in black. Truncated residues of the long SARS-CoV-2 nsp10 construct are highlighted in green, and additional missing residues of the short SARS-CoV-2 nsp10 construct are highlighted in blue. The percent identity between SARS-CoV-2 and SARS nsp10 proteins is 97.1%. In contrast, the percent identity between SARS-CoV-2 and MERS nsp10 proteins is 59.4%. The alignment was carried out using CLUSTAL OMEGA, 1.2.4 [34] while the secondary structure was assigned by DSSP as implemented in ESPript 3.0 [35].

Figure 2

Overlay of crystal structures of nsp10 from MERS, SARS, and SARS-CoV-2. Ribbon diagram of crystal structures of nsp10 MERS (grey, PDB entry 5YN5), SARS (yellow, PDB entry 2G9T), and SARS-CoV-2 (blue, PDB entry 7DIY) with structural zinc atoms shown as blue spheres. View on right is rotated by 180° along the horizontal axis with respect to view on the left. The three nsp10 crystal structures downloaded from the RCSB PDB were superposed in Pymol (v2.4.1) [36].

Figure 3

SDS-PAGE of SARS-CoV-2, SARS and MERS nsp10 constructs purified for this study. Lane designation: MW contains the molecular weight marker proteins (in kDa) and the nsp10 proteins in the other lanes are indicated by names. The faint bands slightly above 70 kDa were identified by mass-spectrometry analysis as chaperone protein DNAK from E. coli (UniProtKB ID: P0A6Y8; calculated MW: 69.1 kDa).

Figure 4

(A) Experimental SAXS data measured for nsp10 proteins from SARS-CoV-2 (short, long and full-length) and the full-length nsp10 proteins from MERS and SARS coloured in light green, dark green, red, blue and yellow respectively in this figure and Figure 5. Data are shown in relative scales. (B) Distance distribution profiles of the scattering data from (A).

Figure 4

(A) Experimental SAXS data measured for nsp10 proteins from SARS-CoV-2 (short, long and full-length) and the full-length nsp10 proteins from MERS and SARS coloured in light green, dark green, red, blue and yellow respectively in this figure and Figure 5. Data are shown in relative scales. (B) Distance distribution profiles of the scattering data from (A).

Figure 5

Comparison between the measured scattering data and the calculated scattering curve from the model (in black and in cartoon representation on the right). Discrepancy χ² is shown on the header of each plot. The nsp10 coordinates were extracted from the PDB.

Figure 6

Superimposition of the full-length ab initio nsp10 SARS-CoV-2 model shown as a molecular envelope with the rigid body/ab initio model of the same protein shown as cartoon in two different orientations. The ab initio envelope was generated in chimera using a map covering the calculated coordinates with a resolution of 13 Å. The extra residues added in the rigid body/ab initio are shown as blue spheres. Maximum dimensions for two of the orientations are indicated on the left as well as the N- and C-terminal boundaries of the model.