2.1. Nsp1 structural analysis using molecular dynamics (MD)
simulation
The Nsp1 protein sequences (Polyprotein1ab) from SARS-CoV-2 (P0DTD1) and
SARS-CoV-1 (P0C6X7) were retrieved from the UniProtKB
(http://www.uniprot.org/). The structure of the Nsp1-ribosome
complex PDB was downloaded from SARS-CoV-2 Proteome-3D[17] (https://sars3d.com/model/nsp1) to
investigate the interaction between Nsp1 and the ribosomal 40S subunit.
The alignment between SARS-CoV-2 and SARS-CoV-1 Nsp1 sequences was
performed using the EMBOSS Needleman–Wunsch method[18,19]. The I-TASSER server[20] was used to build the 3D structure of both
Nsp1 proteins. The best model was selected based on its C-score and the
quality of the predicted structure was confirmed by PROCHECK[21,22] and ProSA [23],
which calculate an overall quality score for 3D structures
(Supplementary Figure S1). The local environment of amino acids was
investigated through the WHAT IF coarse packing quality control[24]. The H++ server was used to predict the
protonation of histidine residues at 7.4 pH [25].
The UCSF Chimera (1.14) was used for visualization and analysis of
structures [26]. The molecular dynamics (MD)
simulation were carried out using the GROMACS 2020.2 software in OPLS
force field. A water box was created with at least 1 nm (10 Å) distances
from the protein using the SPC water model and applying boundary
conditions. The system neutralization was done by adding
Na+ and Cl- ions at the
concentration of 0.1 M. The MD simulation was carried out to examine the
quality of the model structures by investigating their stability via
performing 100 ns simulations at a constant temperature 310 K (NVT).
Energy minimization was performed in 50,000 steps to avoid any bad
contacts generated while solvating the system. Then, the NPT
optimization was done for 100 ps. To increase the likelihood of
achieving the appropriate structure, the MD simulation was performed for
100 ns using the OPLS force field. The MD simulation was performed in
three replicas for SARS-CoV-2 and two for SARS-CoV-1 Nsp1. At the end of
this process, the GROMACS Tools package were used for the trajectory
analysis; including root-mean-square deviation (RMSD), residue-based
root mean square deviation (RMSF), radius of gyration (Rg), solvent
accessible surface areas (SASA), etc. The RMSD and Rg were also applied
to calculate the Free Energy Surface (FES) of SARS-CoV-2 and SARS-CoV-1
Nsp1. The trajectory files of Principal Component Analysis (PCA) were
analyzed by using g_covar and g_anaeig within the GROMACS package.
Eigenvectors of the covariance matrices and the projections of the first
two principal components were computed.