To investigate PPIs between hACE2 and RBD domains of the three coronaviruses, we collected 22 experimental structures summarized in Table 1 and performed FMO-DFTB3/D/PCM calculations for all the experimental structures. Due to structural arrangements from mutations summarized in Table S1, we collected all available structures to consider them together. Subsequently, we performed hot spot analysis using the FMO/3D-SPIEs tool. We used the subscripts ‘HC’ and ‘LC’ to indicate the heavy chain and light chain in the antibody, respectively.
3.1 Hot spot region between hACE2 and RBD-SARS-CoV-1
In order to investigate the hot spot region in the RBD of the SAR-CoV-1 and hACE2 receptor complex, we performed FMO calculations on 12 RBD-SARS-CoV-1/hACE2 complexes (Supplementary Table S2-S13). We summarized the FMO results in Figure 1. In 12 RBD-SARS-CoV-1/hACE2 complexes, the FMO results detected 69 amino acid pairs as the union of the amino acid pair members from all the 12 complex structures. Among the 69 amino acid pairs, all 12 complexes have two common amino acid pairs, 1) a cation-π interaction between K353 in hACE2 and F483 in RBD-SARS-CoV-1, and 2) a hydrogen bond interaction between D355 in hACE2 and T486 in RBD-SARS-CoV-1. Ten of the twelve complexes have a common amino acid pair, an electrostatic interaction between the carboxyl oxygen of K353 and T487 and Y491 in RBD-SARS-CoV-1. Another 10 complexes also have an electrostatic interaction between R357 in hACE2 and T487 in RBD-SARS-CoV-1. Nine of the twelve complexes have six common amino acid pairs in hACE2/RBD: S19/D463, F28/Y475, Q325/R426, E329/R426, K353/Q492, and D355/G488. Eight of the twelve complexes have three common amino acid pairs: Q42/Y484, Y83/N473, and G354/G488. Seven of the twelve complexes have one common amino acid pair: D38/Y436. Six of the twelve complexes have one common amino acid pair: D38/G482.
The amino acid pairs that contributed to the stability of the complexes are well correlated with the published site-directed mutagenesis studies. Wu et al. reported that 2 mutations (D38A and Y41F) in hACE2 and 3 mutations (Y491A, T487A, and T487S) in RBD-SARS-CoV-1 lowered the binding affinity.19 Li et al. reported that 10 mutations in hACE2 affected the inhibition of interactions with the SARS-CoV-1 spike protein (Q24K/K26E, K31D, Y41A, K68D, K353D, K353A, K353D, D355A, R357A, and R393A).20 When comparing the 69 amino acid pairs of this study with the mutagenesis experimental results from two papers, it was confirmed that 32 of the 69 amino acid pairs correlated with the experimental results.
The changes in the binding affinity between the proteins that form a complex by mutation can be explained by comparing the structural changes (i.e. changes in the amino acid pairs that contribute to the increase or decrease of the binding affinity) of the mutated proteins with those of the wild-type proteins. Qu et al. reported that the N479K/T487S mutation on RBD-SARS-CoV-1 lowers the binding affinity.21 One complex (PDB ID: 3D0H) has the T487S mutation in RBD-SARS-CoV-1. T487 in WT RBD-SARS-CoV-1 attractively interacts with 6 amino acids, Y41, G326, N330, G354, F356, and R357, whereas S487 in the mutated complex attractively interacts with only 3 amino acids, N330, G354, and R357. Wu et al. reported that the K31T mutation on hACE2 increases the binding affinity,22 because the K31 in WT hACE2 (PDB ID: 2AJF) interacts only with Y442 of RBD-SARS-CoV-1, whereas T31 in the mutated hACE2 (PDB ID: 3D0G) interacts with two amino acids, Y442 and Y475.
3.2 The common hot spot region in RBD-SARS-CoV-1 against hACE2 and SARS-CoV-1 antibodies
In order to narrow down the hot spot regions between hACE2 and RBD-SARS-CoV-1, we performed FMO calculations on four RBD-SARS-CoV-1/antibody complexes (Supplementary Table S14-S19). We summarized the FMO results in Figure 1.
In RBD-SARS-CoV-1/80R (PDB ID: 2GHW) complex, the FMO results detected 30 amino acid pairs, which are summarized in Supplementary Table S14. The amino acid pairs were in agreement with the results previously reported by Hwang et al.23 When comparing the 30 amino acid pairs of this study with the previously reported results, it was confirmed that 17 of the 30 amino acid pairs are correlated: D426/Y53, Y433/W226, D437/R162, Y440/D182, P470/D202, N479/D182, D480/R162, D480/S163, D480/N164, D480/R223, Y481/R223, Y484/Y102, T486/Y53, T487/Y53, T487/D99, G488/A33, and Y491/D99. In RBD-SARS-CoV-1/m395 (PDB ID: 2DD8) complex, the FMO results detected 18 amino acid pairs, which are summarized in Supplementary Table S15. The amino acid pairs that contributed to the stability of the complexes are well correlated with the published site-directed mutagenesis study, in which the T487 mutation does not significantly affect the neutralizing activity of the antibody.24 The FMO results supported that T487S mutation would change only minor van der Waals interactions between T487 and HCY32. In the RBD-SARS-CoV-1/S230 (PDB ID: 6NB6, 6NB7) complex, the FMO results detected 25 amino acid pairs, which are summarized in Supplementary Table S16-S18. The S230 binds to RBD-SARS-CoV-1 in different two states. The FMO results of state 1 are detailed in Supplementary Table S16, and those of the state 2 are mentioned in Supplementary Table S17-S18. In the RBD-SARS-CoV-1/F26G19 (PDB ID: 3BGF) complex, the FMO results detected 24 amino acid pairs, which are summarized in Supplementary Table S19.
In order to find common hot spot amino acids in RBD-SARS-CoV-1 against hACE2 and SARS-CoV- 1 antibodies, we illustrated the FMO results with a 3D-SPIEs-based map. (see Figure 1). All four antibodies (80R, m395, S230, and F26G19) and hACE2 have two common amino acids, R426 and T487, in RBD-SARS-CoV-1. Three of the four antibodies and hACE2 have four common amino acids, T486, G488, I489, and Y491, in RBD-SARS-CoV-1. Two of the four antibodies and hACE2 have two common amino acids, F483 and Q492, in RBD-SARS-CoV-1. Only S230 and hACE2 share four common amino acids, D463, N473, Y475, and Y442, in RBD-SARS-CoV-1. Only 80R and hACE2 share two common amino acids, Q479 and Y484, in RBD-SARS-CoV-1. Other interactions between antibodies and RBD-SARS-CoV-1 do not share interactions between hACE2 and RBD-SARS-CoV-1. Considering the possibility of mutation prediction in viruses by the FMO methods,25, 26 the evolutionary process of SARS-CoV-1 can be performed to elude neutralization of antibody by switching the unshared interactions between the antibody and hACE2 receptor.
According to the map, there are two hot spot regions between hACE2 and RBD-SARS-CoV-1 (See Figure 1). The first hot spot region on hACE2 receptor consists of D30, K31, and several residues. The counter part of that on RBD-SARS-CoV-1 comprises Y442, D463, N473, N479, and so on. The second hot spot region on hACE2 consists of D38, Y41, K353, D355, and several residues. The counter part of that on RBD-SARS-CoV-1 comprises R426, T486, T487, I489, Y491, and so on. We found that SARS- CoV-1 antibodies focus on the second hot spot to block the formation of amino acid pairs between hACE2 and RBD-SARS-CoV-1.
3.3 Hot spot region between hACE2 and RBD from HCoV-NL63 and SARS-CoV-2
Although the RBD of HCoV-NL63 does not share structural homology with the RBDs of SARS-CoV- 1 and SARS-CoV-2, the three viruses recognize the same hACE2 receptor to invade host cells. In order to investigate the hot spot region between HCoV-NL63 and hACE2, we performed FMO calculations on the HCoV-NL63/hACE2 complex (PDB ID: 3KBH). The FMO results in which 23 amino acid pairs were detected are summarized in Supplementary Table S20. The FMO results were in agreement with the six amino acid pairs (hACE2/RBD-HCoV-NL63) previously reported by Wu et al.:27 D30/S496, H34/G495, H34/S496, E37/Y498, M323/H586, and G354/G537.
In order to find amino acids in hot spot regions in the PPI interface between SARS-CoV-2 and hACE2, we performed FMO calculations on four SARS-CoV-2/hACE2 complexes (Supplementary Table S21- S24), the results of which are summarized in Figure 1. The FMO results detected 37 amino acid pairs as the union of the amino acid pair members from all the four complexes. Among the 37 amino acid pairs, all four complexes have common interactions: 1) seven hydrogen bond interactions (hACE2/RBD): Q24/N487, E35/Q493, E37/Y505, D38/Y449, D38/G496, K353/F497, and G354/G502, 2) an amide-π interaction between G354 in hACE2 and Y505 in RBD-SARS-CoV-2, and 3) four electrostatic interactions (hACE2/RBD): K31/P491, G354/N501, D355/T500, and D355/G502. Three of the four complexes have common interactions between hACE2 and RBD-SARS-CoV-2: 1) six hydrogen bond interactions (hACE2/RBD): F28/Y489, D30/K417, K31/E484, K31/Q493, Y41/Q498, and Y83/N487, 2) a π-π interaction between Y83 in hACE2 F486 and in RBD-SARS-CoV-2, and 3) four electrostatic interactions (hACE2/RBD): S19/G476, S19/S477, Y41/N501, and R357/N501. Two of the four complexes have five common amino acid pairs in hACE2/RBD: D30/L455, D38/Q498, N330/N501, K353/Q498, and K353/G502.
3.4 The common hot spot region between hACE2 and RBD domains from SARS-CoV-1, HCoV-NL63, and SARS-CoV-2
To investigate the common hot spot region on hACE2 against RBDs from the three viruses, and vice versa, we illustrated the FMO results in Figure 1. In the three viruses, all RBDs have common interactions with D30, K31, E37, K353, G354, and D355 in hACE2. SARS-CoV-1 and SARS-CoV-2 have common interactions with the S19, Q24, F28, E35, A36, D38, Y41, Q42, Y83, E329, N330, and R357 in hACE2. Only SARS-CoV-1 and hACE2 share interactions with E23, A25, F32, T324, Q325, G326, and F356, whereas only NL63-CoV and hACE2 share interactions with N33, M323, and F327. The common interactions between SARS-CoV1 and NL63-CoV were H34 and R393 in hACE2. SARS-CoV-1, which belongs to the same class as SARS-CoV-2, shows almost a similar interaction with hACE2, whereas NL63-CoV shows some difference with regard to its interaction with hACE2.
3.5 Hot spot region in hACE2/RBD-SARS-CoV-2 integrated with 3D-SPIEs-based interaction map
We created a 3D-SPIEs based interaction map to find the hot spot regions from the PPI information between hACE2 and RBD-SARS-CoV-2 (see Figure 1 and Figure 2). When comparing the interacting residues between hACE2 and RBD of the three viruses, there are two hot spot regions consisting of shallow grooves on the hACE2 receptor. The first hot spot consists of D30 and K31. The second hot spot is formed by E37, K353, G354 and D355. According to the map, the second hot spot is expected to be the most important hot spot between hACE2 and RBD-SARS-CoV-2. We observed that the first hot spot on hACE2 has interactions with K417, L455, E484, P491, and Q493 in RBD-SARS-CoV-2, whereas the second hot spot has interactions with R403, F497, Q498, T500, N501, G502, Y505, and Q506 in RBD-SARS-CoV-2. The results from the common hot spot region in SARS-CoV-1 antibodies supported the results that the second hot spot region was important for the PPI between RBD-CoV-1 and its antibodies. It can be used to develop antibodies and antiviral agents by using the information of the hot spot regions suggested in this work.