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.