Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro.

Yadav R, Courouble VV, Dey SK, Harrison JJEK, Timm J, Hopkins JB, Slack RL, Sarafianos SG, Ruiz FX, Griffin PR, Arnold E, Sci Adv 8(49):eadd2191 (2022) Europe PMC

SASDP23 – SARS-CoV-2 non-structural protein 7-11 (nsp7-11) polyprotein monomer

Replicase polyprotein 1a
MWI(0) 73 kDa
MWexpected 60 kDa
VPorod 102 nm3
log I(s) 2.98×100 2.98×10-1 2.98×10-2 2.98×10-3
Replicase polyprotein 1a small angle scattering data  s, nm-1
ln I(s)
Replicase polyprotein 1a Guinier plot ln 2.98×100 Rg: 3.5 nm 0 (3.5 nm)-2 s2
(sRg)2I(s)/I(0)
Replicase polyprotein 1a Kratky plot 1.104 0 √3 sRg
p(r)
Replicase polyprotein 1a pair distance distribution function Rg: 3.6 nm 0 Dmax: 15.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Replicase polyprotein 1a DAMMIF model
Synchrotron SAXS data from solutions of SARS-CoV-2 non-structural protein 7-11 (nsp7-11) polyprotein monomer in 20 mM HEPES, 10% glycerol, 500 mM NaCl, 5 mM DTT, pH 7.5 were collected on the BioCAT 18ID beam line at the Advanced Photon Source (APS), Argonne National Laboratory (Lemont, IL, USA) using a Pilatus3 X 1M detector at a sample-detector distance of 3.7 m and at a wavelength of λ = 0.1033 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line co-flow (sheath cell) size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A sample at 4 mg/ml was injected onto a GE Superdex 200 Increase 10/300 column at 20°C. 2500 successive 0.700 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC-MALS-SAXS. Size separation used a 1260 Infinity II HPLC (Agilent Technologies). UV data was measured in the Agilent, and MALS-DLS-RI data by DAWN HELEOS-II (17 MALS + 1 DLS channels) and Optilab T-rEX (RI) instruments (Wyatt Technology). SAXS data was measured in a sheath-flow cell.

Replicase polyprotein 1a (nsp7-11)
Mol. type   Protein
Organism   Severe acute respiratory syndrome coronavirus 2
Olig. state   Monomer
Mon. MW   59.7 kDa
 
UniProt   P0DTC1 (3860-4405)
Sequence   FASTA