Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we per-formed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003–2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003–2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the S glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2.
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Computational Design of Miniproteins as SARS-CoV-2 Therapeutic Inhibitors
A rational therapeutic strategy is urgently needed for combating SARS-CoV-2 infection. Viral infection initiates when the SARS-CoV-2 receptor-binding domain (RBD) binds to the ACE2 receptor, and thus, inhibiting RBD is a promising therapeutic for blocking viral entry. In this study, the structure of lead antiviral candidate binder (LCB1), which has three alpha-helices (H1, H2, and H3), is used as a template to design and simulate several miniprotein RBD inhibitors. LCB1 undergoes two modifications: structural modification by truncation of the H3 to reduce its size, followed by single and double amino acid substitutions to enhance its binding with RBD. We use molecular dynamics (MD) simulations supported by ab initio density functional theory (DFT) calculations. Complete binding profiles of all miniproteins with RBD have been determined. The MD investigations reveal that the H3 truncation results in a small inhibitor with a −1.5 kcal/mol tighter binding to RBD than original LCB1, while the best miniprotein with higher binding affinity involves D17R or E11V + D17R mutation. DFT calculations provide atomic-scale details on the role of hydrogen bonding and partial charge distribution in stabilizing the minibinder:RBD complex. This study provides insights into general principles for designing potential therapeutics for SARS-CoV-2.
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- Award ID(s):
- 2028803
- NSF-PAR ID:
- 10321676
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 23
- Issue:
- 2
- ISSN:
- 1422-0067
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The COVID-19 pandemic was declared due to the spread of the novel coronavirus, SARS-CoV-2. Viral infection is caused by the interaction between the SARS-CoV-2 receptor binding domain (RBD) and the human ACE2 receptor (hACE2). Previous computational studies have identified repurposed small molecules that target the RBD, but very few have screened drugs in the RBD–hACE2 interface. When studies focus solely on the binding affinity between the drug and the RBD, they ignore the effect of hACE2, resulting in an incomplete analysis. We screened ACE inhibitors and previously identified SARS-CoV-2 inhibitors for binding to the RBD—hACE2 interface, and then conducted 500 ns of unrestrained molecular dynamics (MD) simulations of fosinopril, fosinoprilat, lisinopril, emodin, diquafosol, and physcion bound to the interface to assess the binding characteristics of these ligands. Based on MM-GBSA analysis, all six ligands bind favorably in the interface and inhibit the RBD–hACE2 interaction. However, when we repeat our simulation by first binding the drug to the RBD before interacting with hACE2, we find that fosinopril, fosinoprilat, and lisinopril result in a strongly interacting trimeric complex (RBD-drug-hACE2). Hydrogen bonding and pairwise decomposition analyses further suggest that fosinopril is the best RBD inhibitor. However, when lisinopril is bound, it stabilizes the trimeric complex and, therefore, is not an ideal potential drug candidate. Overall, these results reveal important atomistic interactions critical to the binding of the RBD to hACE2 and highlight the significance of including all protein partners in the evaluation of a potential drug candidate.
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Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concernmore » « less
Abstract The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the
N -terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved bothN - andO - glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation. -
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/ijms23020838
