[ad_1]
Using dynamic contact maps and energy differences between the different conformations of the peak protein (or protein S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an international team of researchers recently published a paper on the bioRxiv* preprint server showing the different stable conformations of the peak protein. Determining potential target sites to destabilize the spike protein can help develop drugs that inhibit its binding to host cells.
The spread of coronavirus disease 19 (COVID-19) causes researchers to rush to understand how the disease-causing pathogen SARS-CoV-2 infects and replicates in host cells.
It is established that the spike protein of the virus is key to the entry of the virus into the host cell. It binds to human angiotensin converting enzyme 2 (ACE2) and undergoes many conformational changes. The protein changes the conformation of the receptor-binding domain (RBD) from the bottom up, preparing it to bind to ACE2, and then a subsequent fusion of the viral membrane with the host cell membrane.
Several studies have investigated how changes in the protein conformations of the virus allow it to infect host cells so easily. Computational studies show a correlation between the polybasic furin cleavage site and surface residues in the RBD region that ACE2 recognizes, mediated by electrostatic interactions separated by 10 nm. In the dominant virus D614G mutation, the residues correlated with the mutation are located 7-10 nm from the RBD of SARS-CoV-2.
It has been suggested that these changes could increase its transmissibility and aid in the post-fusion steps with host cells. However, since the spike protein region is generally unstable without the presence of the ACE2 receptor, it is difficult to study the conformation experimentally.
A previous modeling study has demonstrated the presence of a dynamic asymmetry that triggers a change in conformation, with the closed conformation being the ground state.
Representation of the different RBD conformations in the SARS-CoV-2 spike protein. Cell recognition is initiated by the transition of the RBD from the descending to the ascending conformation and then the high affinity triggers the binding between the RBD in the ascending conformation and the ACE2 receptor shown in purple and green respectively. The sequence of a peak protein chain is shown at the top, as well as the residue numbers for various protein domains. The bar shows the typical length scale for the entire system. The fusion of the viral and cellular membrane takes place by means of surface proteases that cleave each chain at the polybasic sites (yellow stars) located at the interface of the S1 / S2 subunits.
Different conformations of RBD
Using computational models, the researchers now show the correlations between the different conformations of the spike protein and its subunits, RBD, and the N-terminal domain (NTD).
Analysis of the differential contact map has revealed how the change in the number of contacts between different conformations affects the stability of a conformation.
The authors found that although the one up and two down RBD conformation had fewer destabilizing residues than the two up and one down conformation, the number of stabilizer residues was similar for the two conformations. The number of destabilizing residues for the one upward RBD and two upward RBD conformation that are formed from the three downward conformation is about 3% and 15% of the total number of residues. This suggests that going to the conformation of an RBD upward results in the destabilization of fewer residues.
When analyzing the high-frequency contacts, the researchers found 25 stabilizing residues in the downward conformation, making 83 contacts that are critical for the 40 contacts made in the case of an upward RBD. Such transitions occur by rearrangements of some residues in the lower part of the RBD. The identification of residues that form stabilizing contacts is important in the development of drugs that target the binding or disruption of viral proteins.
An earlier study identified a free fatty acid that binds to a hydrophobic pocket that blocks the spike protein in the closed conformation. This is possible because there are few native contacts in the two closest RBDs. The authors also found additional contacts between the ascending chain of the RBD and the NTD, which helped stabilize the conformation. In contrast, there were few interactions between the residues in the two RBD chains in the downward conformation.
The upper panels show changes in terms of stability between the closed (3down) and open (1up2down) states. Panel (A) shows the case of RBD in chain B stabilized by neighboring protein domains such as RBD and NTD in chain C and A respectively. The 25 stabilizing residues are highlighted in green and establish several native high frequency (NC) contacts equal to 83. Panel (B) shows the same set of residues from panel (A) that destabilize in the open state forming 40 contacts. Panel (C) shows the stabilization due to 25 contacts between all RBDs in the closed state. The structure of LA (in cyan) has been superimposed on our results, as it was shown to block the closed state by forming contacts between two adjacent RBDs. Panel (D) shows the RBD in ascending conformation that has been stabilized by 19 contacts formed between the NTD and two other RBDs. The positions of two N-glycans that help structurally by making extensive interaction with the RBD in the upward conformation have been superimposed on our structure. We highlight the residue contacts that are responsible for stabilization by means of dashed black lines.
Targeting sites of destabilization can inhibit viral binding
The team also determined the role of electrostatic interactions in the spike protein using the Poisson-Boltzmann method, taking into account the contributions of solvation energy and Coulomb interactions.
They found that the most favorable position is when all RBDs are in the downward conformation. The one RBD up conformation is the most favorable open position, followed by the two RBD up conformation. The latter conformation has a much higher energy barrier compared to changing from the closed position to an up RBD position.
The high frequency contacts between the RBD chain and the NTD chain suggest that they play a role during the transition from the closed to the open state. This transition occurs at an energy cost of approximately 10-15 kcal / mol by breaking the links between these chains.
In the absence of ACE2, a large amount of energy is required; about 30 kcal / mol for the transition from the closed conformations to the two RBD upwards. This suggests that the spike protein is likely to be in the upstream RBD conformation prior to interaction with the host cell.
Information on potential target sites to destabilize the spike protein could be used to investigate potential approved or herbal-derived drugs that can inhibit the spike protein conformations that allow binding to ACE2, the authors write.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.
