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Your Position: Home > COVID-19 R&D > SARS-CoV-2 Spike protein mutation may increase the virus infectivity
SARS-CoV-2 Spike protein mutation may increase the virus infectivity
Release time: 2020-05-04 Source: ACROBiosystems Read: 3384

The Spike protein of SARS-CoV-2 facilitates the viral entry into target cell exploiting Angiotensin-converting enzyme 2 (ACE2) receptor. [1][2] The Spike protein receptor-binding domain (RBD), which is on the S1 subunit of SARS-CoV-2, is the critical determinant of viral tropism and infectivity. The genome analysis of global SARS-CoV-2 strains yields 32 RBD mutant strains clustering into 10 mutation types under high positive selection pressure. Three mutation types circulating in Wuhan, Shenzhen, Hong Kong, and France, displayed enhanced structural stability along with higher human ACE2 receptor affinity of the spike protein, indicating these mutants may have acquired increased infectivity to humans. [3]


SARS-CoV-2 RBD mutation mapping

It is believed that the RBD should be highly conserved because it is the only domain to bind human ACE2 and initiate cell entry. Surprisingly, the RBD sequences were as diverse as the other regions of the S protein, revealed by polymorphism and divergence analysis.

Among the 1609 SARS-CoV-2 strains in the public database, 32 strains contained amino acid mutations in the RBD. These mutant strains were reported from multiple locations including China, the UK, Finland, France, Belgium, the USA, and India, most of which had only one amino acid change compared to the originally reported genome.

1.png

Fig. 1: Distribution of the SARS-CoV-2 strains mutated in the RBD of the S protein. The geographic distribution of the RBD mutant strains is displayed. The strains with names highlighted in red are mutants with the enhanced binding affinity. The strains with names noted in yellow are mutants with similar binding affinities. 

(Source: bioRxiv preprint doi: https://doi.org/10.1101/2020.03.15.991844)


Three Mutation types bind human ACE2 receptor with higher affinityffinity


Molecular dynamics simulations were performed to estimate the functional change suggested by the RBD mutations. Three RBD mutant types (N354D and D364Y, V367F, W436R) exhibited significantly lowered binding free energy (ΔG), suggesting a significantly increased affinity to human ACE2. [3]


Structural basis for increased affinity

The binding surface of the RBD to ACE2 is largely in random coil conformation, which lacks structural rigidity. Therefore, a firm scaffold should help to maintain this conformation of the interaction surface, and thus may facilitate the binding affinity. The beta-sheet structure scaffold, centered by residues 510-524 (Fig. 2, marked as red), provides this rigidity. 


“Higher affinity” mutants (N354D D364Y, V367F, and W436R) exhibited a general decreased ΔG in the binding site region, which increased the structural stability. What’s more, the mutation W436R provides a positively charged Arg in the proximity of the complementing highly negative charged ACE2 surface. This potential electrostatic attraction may contribute to the higher affinity.[3]


1588562225165449.png

Fig. 2: Structural analysis of RBD mutants and the effects on their binding affinity. Spatial 409 location of the mutant amino acids and the fragment 510-524. 

(Source: bioRxiv preprint doi: https://doi.org/10.1101/2020.03.15.991844)


Reference:

1. Hoffmann et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor, 2020, Cell 181, 271–280;

2. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin, 2020, Nature 579, 270-273;

3. Ou et al. Emergence of RBD mutations in circulating SARS-CoV-2 strains enhancing the structural stability and human ACE2 receptor affinity of the spike protein, bioRxiv preprint doi: https://doi.org/10.1101/2020.03.15.991844;


To investigate the findings discussed above, ACROBiosystems expressed the recombinant prototype spike protein RBD and “higher affinity” mutants in house. They used three different methods, including ELISA, Biacore SPR, and Octet BLI, to analyze the binding affinities between human ACE2 protein and different spike proteins respectively. The results were summarized in Fig. 3, Fig. 4, and Fig. 5 below. As you can see, all the “higher affinity” spike protein RBD mutants showed stronger binding affinities compared to the prototype.


Bioactivity-ELISA

1588562283775710.png

Fig. 3: Bioactivity- ELISA results. Human ACE2, Fc Tag (Cat. No. AC2-H5257) was immobilized on the plate. RBD (V367F) EC50: 0.31nM > RBD (W436R) EC50: 0.89nM > RBD (N354D, D364Y) EC50: 0.97nM > prototype RBD EC50: 1.47nM.


Bioactivity-BLI

1588562314902779.png

Fig. 4: Bioactivity – Octet BLI results. The KD between Human ACE2 and prototype spike protein RBD is 24.4nM. The KD between Human ACE2 and RBD(V367F), RBD (W436R), and RBD (N354D, D364Y) were 5.5nM, 6.85nM, and 6.33nM respectively.


Bioactivity-SPR

1588562358198848.png

Fig. 5: Bioactivity – Biacore SPR results. The KD between Human ACE2 and prototype spike protein RBD is 13.1nM. The KD between Human ACE2 and RBD(V367F), RBD (W436R), and RBD (N354D, D364Y) were 4.33nM, 6.96nM, and 7.57nM respectively. 


Product List

Kits  For research use only.
MoleculeCat. No.SpeciesProduct DescriptionOrder/Preorder
S protein RBDEP-105SARS-CoV-2SARS-CoV-2 Inhibitor Screening Kit

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S1 proteinMBS-K001SARS-CoV-2SARS-CoV-2 Spike S1 protein-coupled magnetic beads

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S protein RBDMBS-K002SARS-CoV-2SARS-CoV-2 Spike protein RBD-coupled magnetic beads

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S1 proteinTAS-K001SARS-CoV-2SARS-CoV-2 antibody titer assay kit (Spike protein S1)

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S protein RBDTAS-K002SARS-CoV-2SARS-CoV-2 antibody titer assay kit (Spike protein RBD)

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Antibody
MoleculeCat. No.SpeciesProduct DescriptionOrder/Preorder
S protein RBDSAD-S35HumanAnti-SARS-CoV-2 neutralizing antibody (Human IgG1)

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Proteins
MoleculeCat. No.SpeciesHostProduct DescriptionOrder/Preorder
ACE2AC2-H82E6HumanHEK293Biotinylated Human ACE2 / ACEH Protein, His,Avitag™ (MALS verified)

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AC2-H82F9HumanHEK293Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag™

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AC2-H5257HumanHEK293Human ACE2 / ACEH Protein, Fc Tag (MALS verified)

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AC2-C52H7CynomolgusHEK293Cynomolgus ACE2 / ACEH Protein, His Tag

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AC2-H52H8HumanHEK293Human ACE2 / ACEH Protein, His Tag (MALS verified)

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AC2-R5246RatHEK293Rat ACE2 / ACEH Protein, His Tag (MALS verified)

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AC2-M5248MouseHEK293Mouse ACE2 / ACEH Protein, His Tag (MALS verified)

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AC2-P5248Paguma larvataHEK293Paguma larvata ACE2 / ACEH Protein, His Tag

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S proteinSPN-C52H4SARS-CoV-2HEK293SARS-CoV-2 (COVID-19)  S protein (R683A, R685A), His Tag

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SPN-C52H8SARS-CoV-2HEK293SARS-CoV-2 (COVID-19)  S protein (R683A, R685A) , His Tag, active trimer (MALS verified)

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SPN-S52H5SARSHEK293SARS S protein (R667A), His Tag

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S1 proteinS1N-C52H3SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S1 protein, His Tag

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S1N-C82E8SARS-CoV-2HEK293Biotinylated SARS-CoV-2 (COVID-19) S1 protein, His,Avitag™ (MALS verified)

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S1N-S52H5SARSHEK293SARS S1 protein, His Tag (MALS verified)

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S1N-C52H4SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S1 protein, His Tag (MALS verified)

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S1N-C5255SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S1 protein, Fc Tag

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S1N-C5257SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S1 protein, Mouse IgG2a Fc Tag

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S protein RBDSPD-C82E9SARS-CoV-2HEK293Biotinylated SARS-CoV-2 (COVID-19) S protein RBD, His,Avitag™ (MALS verified)

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SPD-C5255SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD, Fc Tag (MALS verified)

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SPD-S52H6SARSHEK293SARS S protein RBD, His Tag (MALS verified)

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SPD-C52H3SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD, His Tag (MALS verified)

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SPD-C5259SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD, Mouse IgG2a Fc Tag

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SPD-S52H4SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD (V367F), His Tag

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SPD-S52H5SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD (N354D), His Tag

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SPD-S52H7SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD (W436R), His Tag

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SPD-S52H8SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD (R408I), His Tag

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SPD-S52H3SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S protein RBD (N354D, D364Y), His Tag

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Nucleocapsid proteinNUN-C51H9SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

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NUN-C5227SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

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NUN-C81Q6SARS-CoV-2E.coliBiotinylated SARS-CoV-2 (COVID-19) Nucleocapsid protein, His,Avitag™

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Envelope proteinENN-C5128SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) Envelope protein, His Tag

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Papain-like ProteasePAE-C5148SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) Papain-like Protease Protein, His Tag

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NSP1NS1-C51H7SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP1 Protein, His Tag

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NSP7NS7-C51H6SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP7 Protein, His Tag

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NSP8NS8-C5149SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP8 Protein, His Tag

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NSP7 & NSP8NS8-C5125SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP7&NSP8 Protein, His Tag

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ACE2AC2-C5245CanisHEK293Canis ACE2 / ACEH Protein, His Tag

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S2 proteinS2N-C5253SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S2 protein, Fc Tag

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S2N-C5254SARS-CoV-2HEK293SARS-CoV-2 (COVID-19) S2 protein, Mouse IgG2a Fc Tag

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NSP15NS5-C5145SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP15 Protein, His Tag

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Expected launch data: May 18

NSP16 & NSP10NS0-C51W3SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP16&NSP10 Heterodimer Protein, His Tag&Twin Strep Tag

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Expected launch data: May 18

NSP14NS4-C5144SARS-CoV-2E.coliSARS-CoV-2 (COVID-19) NSP14 Protein, His Tag

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Expected launch data: May 12

TMPRSS2TM2-H5243HumanHEK293Human TMPRSS2 Protein, His Tag

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