Wed. Aug 17th, 2022

The Corona disease 2019 (COVID-19) pandemic disrupted the normal functioning of society, business and education while taking a serious toll on human health. The pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has undergone several rounds of mutations to emerge as a multitude of variants.

Study: Site Density Functional Theory and Structural Bioinformatics Analysis of SARS-CoV Spike Protein and hACE2 Complex.  Image credit: Juan Gaertner / ShutterstockStudy: Site Density Functional Theory and Structural Bioinformatics Analysis of SARS-CoV Spike Protein and hACE2 Complex. Image credit: Juan Gaertner / Shutterstock

Given the serious threat posed by successive coronavirus, researchers have examined the binding between the angiotensin-converting enzyme 2 (ACE2) molecule, which acts as the virus receptor on the target host cell, and the virus receptor binding domain (RBD) on its tip protein. A new article in the journal Molecules describes this in molecular terms and compares SARS-CoV-2 with a former highly pathogenic coronavirus, SARS-CoV.


The nail protein consists of a homotrimer in the natural state, where each protomer has an S1 and an S2 subunit. The S1 subunit contains RBD, which binds the ACE2 peptidase domain and helps stabilize the tip in its prefusion conformation. To better understand this process, the researchers in the current paper used a method based on density functional theory (DFT).

The application of DFT to biological macromolecules is a complicated affair due to the dimensional nature of these molecules, meaning that six-dimensional density profiles are needed to study rigid molecules with DFT. One potential solution is site density representation, which refers to the density of each atom in a solvent molecule, based on which site density functional theory (SDFT) was developed.

This is suitable for any study that requires structural analysis at the atomic level, as it looks at the density of individual atoms and chemical bonds. However, chemical bonds are localized through strong interactions, in contrast to the equally important long-range but weaker intermolecular forces.

It is difficult to apply SDFT to both types of forces simultaneously, which is a major challenge in the use of this method for molecular fluids. The three-dimensional reference interaction site model (3DRISM) is a simplified SDFT version that is a partial answer to this problem, by representing such liquids as an “atomic” mixture, thus paving the way for the use of more simple liquid theory techniques.

With coronavirus, some other problems arise, such as the large size of the virus and the low degree of flexibility of the virus structure. To reduce the cost of evaluating such structures using conventional mechanics with 3DRISM, the researchers used this study instead of structural bioinformatics. This includes techniques such as molecular docking, studies of affinity and binding constraints, limited geometric simulations (CGS) to sample different conformations, and normal state analysis (NMA).

The use of 3DRISM allowed researchers to study the interactions between the protein and its ligands, in addition to environmental effects due to electrostatic interactions, hydrogen bond formation, steric collisions, and van der Waals bonds.

3DRISM appears to be an appropriate methodological choice to reveal a role of electrostatic and short-range protein-solvent interactions in solvation thermodynamics, as the theory can be used to detect water-mediated contacts in protein. “

What did the study show?

The researchers examined the tip protein protoms of SARS-CoV and SARS-CoV-2 isolated as well as RBD-ACE2 formed in both cases. An important difference between the two is the RRAR insertion at the furin recognition site in the SARS-CoV-2 peak at the S1 / S2 interface compared to the individual arginine in SARS-CoV.

The RBD moves like a hinge with this change in conformational motion before it recognizes the host receptor. For both viruses, at least one RBD must be in the “up” conformation for ACE2 binding, otherwise the ACE2 peptidase will collide with the tip of the three-down conformation.

With SARS-CoV-2, however, the area covered by RBD is larger due to a conformational change. The binding backbone is more compact, and virus-binding hotspots at the interface are more stable due to changes in the amino acids at the interface. This allows for greater interactions between the molecules.

The molecular interactions occur via loops and beta sheets in RBD versus one alpha helix of ACE2, indicating inherent instability. Most interactions happen at the two ends and in the middle. Calculation modeling shows comparable binding-free energy for both RBD-ACE2 complexes, but experimental evidence indicates that SARS-CoV-2 RBD-ACE2-binding-free energy is lower than the other by about 1.1 kcal / mol.

While the chemical shifts between the two complexes are negligible, the relative absolute differences at the interface are marked, especially near residues that form hydrogen bonds. When water-mediated interactions are also considered, using only water molecules at the most likely positions, strong interactions with both RBD and ACE2 are seen. These were predicted to be stronger for the SARS-CoV-2-RBD-ACE2 complex.

Protein-solvent interaction energies confirmed this shift, with a large decrease in the electrostatic energy leading to a greater improvement in the overall interaction energy of the CoV-2-ACE2 complex. This can be attributed to a higher degree of polarization of the interface water with this complex and the formation of two more water bridges than with CoV-1-ACE2. The water bridges are also stronger with the former complex.

The bridging water molecules form hydrogen bonds with both RBD and ACE2 simultaneously in the former complex, but this is both much weaker and more diffuse with the CoV-1-ACE2 complex. “Therefore, we conclude that bridge water molecules play a significant and perhaps crucial role in the stabilization of the CoV-2-hACE2 complex. “

The binding energies were also predicted to follow the same trend. Yet the difference is always smaller than expected from the experimental data, probably due to the ionic bonds that interfere with the electrostatic and relaxation-induced entropy changes. The results of each method vary considerably.

In addition, several factors such as the state of conformation and the angle of conformation of the molecules in the complex contribute to the binding energy and binding affinity, indicating the need for further studies to validate the 3DRISM results.

Other computational modeling methods suggest that the bound and unbound RBD show large conformational differences for the two viruses. Following this with the NMA, the researchers observed that pairs of residues are linked together. Both RBDs show large degrees of motion in the unbound state, but less for the S2 subunit of the tip.

With ACE2 binding, anti-phase dynamic motions of the expanded RBD region disappear, indicating a stabilization of the molecule with slightly faster RBD motions with SARS-CoV-2.

Again, the SARS-CoV-2 molecule is more rigid than SARS-CoV in the RBD “up” conformation, with the latter showing a larger shift with increasing temperatures. With the latter, two-up and three-up RBDs are common in the unbound state, but are rare with the former. Such findings may underlie the increased binding of RBD-ACE2 to the former.

Overall, the increased strength of interface contacts draws the stability of the bound RBD with SARS-CoV-2. An analysis of the stiffness of the structure also confirms that as the hydrogen bond energy cutoff increases, SARS-CoV loses stiffness but not SARS-CoV-2. This is because the RBD of SARS-CoV becomes a large conformational ensemble with increasing cut-off H-binding energy.

As a result, it loses binding affinity for ACE2. In contrast, the second RBD is mostly a single rigid cluster, which remains rigid on ACE2 binding and favors the proteolytic cleavage leading to membrane fusion.

What are the implications?

The results show how important water is in the RBD-ACE2 interactions, stabilize it and make the CoV-2-ACE2 complex more stable than the CoV-ACE2 complex. The dissociation constant of the latter is an order of magnitude lower than the former.

The lower conformational stability of SARS-CoV RBD compared to SARS-CoV-2 contributes to the lower binding affinity when coupled with the larger surface area of ​​the latter and the large local conformational shifts in the residues. This leads to more RBD-ACE2 interactions, including more hydrogen bonding.

The increased stiffness of the CoV-ACE2 complex compared to that formed by SARS-CoV is due to stronger bridging interactions via water molecules along with two added polar interactions. The binding of the receptor predisposes the molecule to proteolysis and membrane fusion, which favors virus penetration.

Understanding prefusion conformational dynamics as well as its binding mechanism to the receptor among closely related species is essential to design vaccine and inhibitors to stop viral penetration. “


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