S protein amino acid variants

- mutation sites are distributed over the whole length of the SARS-CoV-2 spike protein sequence
- maximum mutation density was near the protease cleavage site
- D614G mutation is the only most common mutation in spike protein so far among various regions
- some mutations at the interface of the spike protein and ACE2 receptor could affect the efficacy of vaccines and other drugs


State University of New York at Stony Brook

The spike glycoprotein (S) is responsible for attachment between the virus and a cell-surface receptor, leading to the fusion of virus to the cell membrane. This membrane-fusion activity can only occur with the proteolytic cleavage of the S glycoprotein into S1 and S2 subunits, which continue to remain associated with one another after cleavage. 


Spike glycoprotein (S)

Guruprasad, L. (2020). Human SARS CoV‐2 spike protein mutations. Proteins: Structure, Function, and Bioinformatics.
https://doi.org/10.1002/prot.26042

Human SARS CoV‐2 spike protein mutations

Using cry-electron microscopy (cryo-EM), the structure of a furin protease-cleaved SARS-CoV-2 S glycoprotein was visualized. These particles were in three different types of conformations: closed, intermediate, and open with an upright receptor-binding domain (RBD). 

Structure of protease-cleaved SARS-CoV-2 S glycoprotein

The presence of a furin-cleavage is favored for infection of SARS-CoV-2 as the cleavage allows the open conformation, exposing the receptor-binding domain (RBD) for easier binding access to a human receptor. The study found that the lower number of S proteins are in a closed conformation for protease-cleaved. There is also an increasing percentage of intermediate form, which is possibly transient, (39%, compared to 34% being closed and 27% being open) demonstrating a high probability that it will shift to an open form and allow binding to a receptor. Therefore, protease cleavage is favored not only for membrane fusion but also for better binding to a receptor by increasing the proportion of S protein on the virus surface. 

Furin protease-cleaved S protein is preferred for SARS-CoV-2

- Cry-electron microscopy (cryo-EM) structures of S glycoprotein of bat virus RaTG13 and uncleaved SARS-CoV-2
- S glycoprotein sequences of RaTG13 and SARS-CoV-2
- S glycoprotein stability 
- Bat virus RaTG13 and SARS-CoV-2 S glycoprotein affinities to human receptor ACE2

Comparison of S glycoprotein between RaTG13 and uncleaved SARS-CoV-2

SARS-CoV-2 relies on its spike (S) protein to bind to cellular receptors to enter host cells. SARS-S, the SARS spike protein, uses ACE2 as an entry receptor into cells. Then the virus utilizes the serine protease TMPRSS2 found within the cell to enhance entry and prime future S proteins. SARS‐CoV‐2 is thought to be more transmissible than others CoVs because its S proteins have a greater affinity for ACE2.

ACE2 as a Receptor for SARS-CoV-2

Partridge et al. have observed multiple heparin binding sites on the SARS-CoV-2 S protein, including:

- one at the furin cleavage site between the S1 and S2 subunits
- others in the S2 domain 
- others in the receptor binding domain (RBD)

SARS-CoV-2 S Protein May Have Multiple Heparin Binding Sites

Prefusion stabilization of the S glycoprotein increases the recombinant expression of viral fusion glycoproteins. Prefusion-stabilized proteins are superior immunogens and can be used in subunit vaccine development. Four substitutions were designed and tested for the SARS-CoV-2 S glycoprotein to produce a more stable construct based on previous knowledge of class I viral fusion protein function.

Prefusion Stabilized SARS-CoV-2 Spike Proteins

- overall low homology between SARS-CoV-2 S Protein and SARS-CoV S Protein
- amino acid sequence identity of the spike proteins is 76.47%
- patches of sequences in RBD show high homology
- amino acid residues at positions 442, 472, 479, 487, and 491 are key to human transmission of the virus
- only Tyr491 is conserved in SARS-CoV-2 S protein; the remaining 4 residues are replaced by other residues of similar properties
- therefore, because the structure of the RBD of SARS-CoV-2 S protein remained the same as SARS-CoV S protein, ACE2 binding is possible and SARS-CoV-2 S protein actually binds with higher binding affinity than SARS-CoV S protein

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