The study of the binding mode of SARS-CoV-2 S protein in complex with ACE2 of Chiroptera may offer important information on the possibility that these species could be a reservoir for the virus. Using ExPASy SWISS-MODEL and having as a template the crystallographic coordinates of hACE2 (PDB ID: 6M0J) [9] we calculated the homology models of the ACE2 proteins presenting not less than 70% sequence homology with hACE2 (Figure 2B). These were subjected to molecular docking against SARS-CoV-2 S protein, imposing as restraints the distances with K31 and K353, that are the most critical residues for S recognition. The data resulting from molecular docking indicates similar binding stability for all the ACE2/SARS-CoV-2 S protein complexes (see Supplementary Table S2). Analysis of the structural models evidences the conservation of the residues within the binding site, highlighting a critical similarity of hACE2 with the ACE2 of the Chiroptera species. In particular, we focused on ACE2 of Myotis daubentonii and Rhinolophus ferrumequinum. These are the solely ACE2 sequences retrieved from the UniProt database that belong to Chiroptera species living in Italy. The homology models of ACE2 belonging to Chiroptera species in complex with S protein were subjected to 1 ns molecular dynamics simulations in water using protein S/hACE2 complex (PDB ID: 6M0J) as control.
Figure 2 (A) Rhinolophus ferrumequinum and Myotis daubentonii ACE2 in complex with SARS-CoV-2 S RBD. The structures are superimposed to hACE2 structure in complex with SARS-CoV-2 S RBD. The data, extracted from the last step of molecular dynamics, are relative to 1–358 ACE2 residues. (B) Multiple sequence alignment including hACE2 and ACE2 of Rhinolophus ferrumequinum and Myotis daubentonii. Residues involved in the interaction with S protein are shown: the residue numbers in red represent the residues essential for hACE2/SARS-CoV-2 S binding. Conserved residues are highlighted in blue, while similar residues are highlighted in green. Bold black residue numbers indicate amino acids essential for the stability of hACE2/SARS-CoV-2 S interaction.