Interaction of sex steroid hormones and dexamethasone with coronavirus protease and spike protein
The three-dimensional geometry of the ligand (steroid molecule)–receptor (coronavirus proteins) complex was obtained from the best docking coordinates (Fig. 1). The chemical structures of DEX and sex steroids T, P4 and E2 are shown in Fig. S1.
The binding energy value for the interaction of E2, P4, T and DEX with coronavirus protease and spike protein is shown in Table 1. Data showed that these steroid molecules had a strong binding affinity to coronavirus protease and spike protein. The binding energy values ranged from -5.9 (4th and 5th T to spike protein) to -8.5 kcal/mol (5th E2 to the main protease) and strongly verified the interaction region between steroid molecules and coronavirus protease and spike protein (Table 1).
Compared to the primary male sex hormone T, the primary female sex steroid hormones E2 and P4 were more likely to interact with coronavirus spike protein (mean ± SD, -6.34 ± 0.54, -6.88 ± 0.43, and -6.88 ± 0.59 kcal/mol found for T, P4 and E2, respectively). Regarding the interaction between sex steroid hormones and the spike protein, the binding energy decreased from -7.1 to -7.8 kcal/mol (~10%) with an increase in E2 (Table 1). The binding energy increased from -7.2 to -5.9 kcal/mol (~ 18%) with an increase in T. For an increase in P4, the binding energy went up from -7.4 to -6.3 kcal/mol (~ 17.5%).
The female hormone E2 had the greatest tendency to interact with the protease enzyme (mean ± SD, -6.74 ± 0.43, -7.12 ± 1.42, and -7.40 ± 0.73 kcal/mol found for T, P4 and E2, respectively). In the case of the interaction between sex steroid hormones and the main protease, the binding energy decreased from -6.5 to -7.5 kcal/mol (~ 15.4%), with an increase in T (Table 1). The binding energy decreased from -7.2 to -7.7 kcal/mol (~ 6.9%) with increasing number of P4. For an increase in E2, the binding energy fell from -7.0 to -8.5 kcal/mol (~ 21.4%, Table 1).
Compared to sex steroid hormones, the value of binding energy was the minimum for DEX, (mean ± SD, -7.64 ± 0.23 and -7.11 ± 0.46 kcal/mol found for the interaction with the main protease and spike protein, respectively). In the case of the interaction between DEX and the main protease, the binding energy decreased from -7.5 to -7.7 kcal/mol (~ 2.7%), with an increase in DEX (Table 1). Regarding the interaction between DEX and spike protein, the binding energy went up from -7.8 to -6.7 kcal/mol (~ 14.1%), with an increase in DEX.
In order to analyze the distribution of amino acids at binding sites, docking data on residues involved in protein-steroid molecule interfaces was conducted. As seen in Table 2, the total number of residues involved in the interaction between steroid molecules and proteins was 117, including ~ 94.9 % (111/117) uncharged and ~ 5.1 % (6/117) charged residues. Moreover, the contribution of aromatic residues (Trp, Phe, and Tyr) was ~ 29.9 % (35/117).
Steroid molecules change the interaction of spike protein with ACE2
The binding free energy and the average hydrogen bond (H-bond) found between the spike protein and ACE2 are shown in Table 3. The calculated interaction energy of the spike protein with ACE2 was -2511.4kJ/mol, which clearly indicated a remarkable affinity (Table 3A). Steroid molecules appeared to be effective ligands in this system, due to an increase in the energy of binding. Compared to the basal interaction (spike protein-ACE2 interaction), the interaction of only five molecules of each steroid with spike protein increased the energy by 201.2 kJ/mol (8%), 192.4 kJ/mol (7.7%), 532.1 kJ/mol (21.2%), and 582.4 (23.2%) for T, P4, E2, and DEX, respectively (Table 3B, C). The H-bond analysis verified the devastating role of steroid molecules in interaction between spike protein and ACE2 (Table 3). The average H-bond over the last 10-ns MD simulation showed a sharp decrease with the docking of five molecules of T (53.6%), P4 (27.5%), E2 (69.5%), or DEX (69.4%) to spike protein before the interaction of spike protein and ACE2.
In order to conduct a computational analysis of the spike protein/ACE2 binding site, a final snapshot of each complex was obtained from a 50 ns molecular dynamic (MD) simulation. As a result, the orientation of the spike protein closing to ACE2 was strongly dependent on the type of steroid molecules (Fig. 2). The important atoms and residues involved in the interaction are shown in Figures S2- S5. On the basis of the Ligplot analysis (Figures S2-S5), the number of effective residues in the spike protein-ACE2 complex binding site and the spike protein-5P4-ACE2 complex binding site was the highest compared to the other systems. In addition, the type of amino acids involved in binding interactions differed in all systems. As an example, Asp12 interacted as a negative charged amino acid of ACE2 (chain A) with Lys85 as a positive charged amino acid of spike protein (chain B) in the absence of hormonal influence. Upon docking of five E2 molecules, these amino acids were not selected as effective amino acid at the binding site. In the presence of five T molecules, Asp12 was bound to Arg76 as a positive charged amino acid of spike protein. This finding indicated that the binding site characteristics of spike protein were altered in the presence of steroid molecules.
In order to analyze the changes in the structure of the spike protein-ACE2 complex upon steroid hormone binding, the solvent accessible surface area (SASA) of the two proteins in the last 10-ns MD simulation was calculated and shown in Fig. 3A. The lowest SASA value was achieved for the spike protein-ACE2 complex compared to other complexes; more surface area of the complex was involved in the spike protein/ACE2 complex interaction. The low SASA value indicated a reduction in the number of water molecules covering the protein surface (Fig. 3B).
Compared to the basal interaction (spike protein-ACE2 interaction), the interaction of 5 molecules of DEX with spike protein increased the interaction energy between spike protein and ACE2 by 582.3 kJ/mol (23.2%, Table 3C). Also, the interaction of 5 DEX molecules with ACE2 increased the interaction energy between spike protein and ACE2 by 239.7 kJ/mol (9.5%) compared to the basal interaction (spike protein-ACE2 interaction, Table 3C). Binding of 5 DEX to spike protein before interaction with ACE2 could widen a considerable gap between spike protein and ACE2, as SASA values increased (Fig. 3C). However, the SASA was not dramatically changed in the case of binding DEX to the ACE2 and to the complex of spike protein-ACE2 complexes (i.e., spike protein /(5DEX-ACE2) or (spike protein-ACE2)/5DEX).
Radius of gyration (RG) of both spike protein and ACE2 indicated that docking T and P4 could make proteins more folded; however, E2 had a different effect (Fig. 3D). The spike protein/ACE2 complex tended to be more unfolding than the complexes in the presence of T and P4. The spike protein-5DEX-ACE2 complex changed the 3-D structure of the spike protein-ACE2 complex to be more unfolded (Fig. 2, Fig. 3E). By contrast, binding DEX to ACE2 or the complex made proteins folded. The result of SASA and RG verified the effect of steroid molecules on the spike protein/ACE2 complex structure.
In addition, the radial distribution function (RDF) analysis (the probability of spike protein in the distance r from ACE2) showed the strongest binding for spike protein/ACE2 complex (Fig. 3F), while both spike protein-5E2-ACE2 ACE2 (Fig. 3F) and spike protein-5DEX-ACE2 complexes (Fig. 3G) had the weakest binding.
Combination of steroid hormones and dexamethasone, and their effect on the interaction of spike protein to ACE2
The three-dimensional geometry of the ligand (five steroid molecules, including E2, P4, T and DEX)-receptor (spike protein-ACE2 complex) is shown in Fig. 2. The changes in the interaction energy between the spike protein and ACE2 in the presence of 5 sex steroid hormones and 5 DEX were shown in Table 3C. As seen in Table 3C, the interaction energy between spike protein and ACE2 was the highest in the presence of DEX along with E2 compared to P4 and T (-994.3, -2009.1, and -2173.4 5 kJ/mol for E2, P4, and T). This indicated a sharp increase in the interaction energy of spike protein and ACE2 in the presence of DEX and E2 by 1517.1 kJ/mol (60.4%), compared to the basal interaction (spike protein-ACE2 interaction).
On the basis of the Ligplot analysis (Figures S6 – S10), it can be clearly seen that the attachment of steroid molecules to spike protein, ACE2, or their complex altered the orientation and residues involved in the interaction. In absence of steroid molecules, nine important residues of ACE2 play a pivotal role in the interaction. Even though, the interaction of five steroid hormones and five DEX molecules reduced the number of residues to 3 and 6 for 5P4 + 5DEX and 5T + 5DEX, respectively. Also, the type of residue-residue interaction changed totally. As an example, Lys335 of ACE2 interacted with Gly164 and Gly170 of spike protein, but in the case of adding 5T + 5DEX, Lys335 interacted with Tyr121 of spike protein (Fig. S10). It should be noted that Ligplot analysis could not detect residues involved in the interaction when 5E2 and 5DEX were included; this was due to a large space that these molecules created between spike protein and ACE2.
Combination of steroid hormones and dexamethasone, and their effect on the structure of spike protein-ACE2 complex
In terms of the data of H-bond average, the number of h-bond plunged by means of docking of 5T + 5DEX (~ 64.8%), 5P4 + 5DEX (~ 62.4%) and 5E2 + 5DEX (~ 86.1%), as shown in Table 3D. The result of SASA clearly indicated that 5 sex steroid hormones and 5 DEX modified the 3-structure of the spike protein-ACE2 complex, while the 5E2+5DEX combination outperformed the other systems (Fig. 4A). The RG analysis clarified a sharp change in the structure of the spike protein-ACE2 complex upon interaction with a combination of 5E2 + 5DEX, causing the complex to be more unfolded (Fig. 4B). However, there was no major alteration in the structure of the complex after interaction with other systems (Fig.4B). In addition, the RDF analysis proved the adverse effect of the combination of sex steroid hormones and DEX on the spike protein/ACE2 interaction (Fig. 4C). With regard to 5E2 + 5DEX combination, there was less probability of spike protein atoms at a distance of r from ACE2, indicating a decreased affinity of spike protein to ACE2 (Fig. 4C).