PMC:7643666 / 6654-11717
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7643666","sourcedb":"PMC","sourceid":"7643666","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7643666","text":"2 Methods\n\n2.1 Construction of homology models\nHomology models of residues 1–358 of Chiroptera ACE2 proteins and bat coronavirus spike protein were built using ExPASy SWISS-MODEL [23, 24]. The templates were chosen according to i) sequence similarity; ii) global model quality estimate (GMQE) value [24]. GMQE is a quality estimation method combining properties from the target–template alignment and the template search method. GMQE value is expressed as a number between 0 and 1: the higher the value, the higher the reliability; iii) qualitative model energy analysis (QMEAN) value [25, 26]. QMEAN is a composite estimator based on different geometrical properties that provide global and local absolute quality estimates based on one single model. The PDB models generated were used for the molecular docking calculation.\n\n2.2 Molecular docking\nMolecular docking was performed using HDOCK server (http://hdock.phys.hust.edu.cn/), which calculates protein-protein interaction through a hybrid algorithm of template-based and template-free docking. The crystal structure of S protein receptor binding domain (RBD) (PDB ID: 6M0J), corresponding to the residues 437–508 of S protein, was docked against the homology models of bat ACE2 [27]. The calculation was carried out imposing as constraints 8 Å distance between S protein A475 and ACE2 E23 and 5 Å distance between S protein N501 and ACE2 D355. The results were analyzed according to i) docking score value (kcal/mol), generated by HDOCK scoring function [28]; ii) ligand root-mean-square deviation (RMSD) of atomic positions value (Å). RMSD measures the distance between the docked pose and a model of the same ligand predicted by template-based homology modelling generated by HDOCK (the lower the value, the more similar the poses).\n\n2.3 Molecular dynamics\nThe structures resulting from molecular docking simulation were subjected to 1 ns molecular dynamic (MD) simulations using GROMACS [29, 30], (Gromos96 53a6 force field) [31]. The structure was immersed in explicit water using the SPC model [32]. The protein was solvated; the system was neutralized by adding Na+ ions, energy minimized, and equilibrated using NVT and NPT runs. The temperature and pressure were kept constant at 300 K and 1.01325 bar using the Berendsen weak coupling-method [33]. The results were used for an MD simulation using Particle Mesh Ewald for long-range electrostatics under NPT conditions. Coordinates were saved every 100 ps. Trajectory files containing the structural coordinates of receptor-ligand complex sampled every 100 ps were fitted in the box and converted in PDB coordinates using the trjconv tool of GROMACS Package. The structures were visualized with Maestro by Schrödinger [34] (see Supplementary Figures S1 and S7).\n\n2.4 Analysis of ACE2 sequences of human and Chiroptera species present in Italy\nAmino acid sequences of Chiroptera ACE2 living in Italy were retrieved from UniProt database. The sequences of Rhinolophus ferrumequinum (UniProt ID: E2DHI2 and B6ZGN7) and Myotis daubentonii (UniProt ID: E2DHI8), were aligned to hACE2 sequence (UniProt ID: Q9BYF1) using Clustal Omega [35, 36]. Residues 1–358, including all the amino acids interacting with S protein RBD, were considered for alignment.\n\n2.5 Analysis of ACE2 sequences of wild and domestic animal species present in Italy\nSequence similarity search over domestic and wild animal ACE2 sequences was carried out using as template 1–358 ACE2 residues of Rhinolophus ferrumequinum and Myotis daubentonii. ACE2 sequences of species not living in Italian territory were excluded. ACE2 sequences of domestic and wild animals with distribution areas on the Italian territory were selected if presenting sequence homology not less than 70%.\n\n2.6 Analysis of human SARS-CoV-2 S and spike protein of Bat coronavirus Rp3\nSequence alignment was carried out using the RBD (residues 339–490) of bat coronavirus Rp3/2004 S protein (UniProt ID: Q3I5J5), and residues 336–518 of SARS-CoV-2 S protein (UniProt ID: P0DTC2). The alignment was carried out using Clustal Omega [35, 36] (see Table 1).\nTable 1 ACE2 sequences of wild and domestic species present in Italy with their UniProt codes and the percentage of identity with human and ACE2 of Chiroptera species present in Italy.\nWild and domestic species present in Italy UniProt ID % Identity with hACE2 % Identity with M. daubentonii ACE2 % Identity with R. ferrumequinum (E2DHI2) ACE2 % Identity with R. ferrumequinum (B6ZGN7) ACE2\nEquus caballus (Horse) F6V9L3 84.72 82.40 84.36 84.92\nFelis catus (Cat) Q56H28 83.06 80.44 82.12 82.68\nOryctolagus cuniculus (Rabbit) G1TEF4 83.90 81.84 79.33 79.89\nUrsus arctos horribilis (Grizzly bear) A0A3Q7TE16 82.22 78.49 80.16 80.73\nCanis lupus familiaris (Dog) A0A5F4BS93 80.56 77.65 78.49 79.05\nVulpes vulpes (Red fox) A0A3Q7RAT9 80.27 77.93 79.05 79.61\nMus musculus (Mouse) Q8R0I0 80.83 78.61 78.71 79.33\nSus scrofa (Pig) K7GLM4 80.55 77.34 77.93 78.49\nBostaurus (Cattle) Q58DD0 78.67 76.19 78.49 79.05\nOvis aries (Sheep) W5PSB6 79.10 75.98 78.27 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