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An allosteric center or site is where a molecule other than the protein substrate can bind, changing the conformation of the protein. An article recently uploaded to the pre-print server bioRxiv* by researchers at Chapman University in California (February 22, 2021) used computer methods to screen for the affinity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants with the enzyme receptor converting angiotensin 2 (ACE2) and monoclonal antibodies REG10987 and REG10933. The analysis allowed the group to identify allosteric centers that provide long-distance communication within the protein. The group suggests that it is these centers that allow the SARS-CoV-2 spike protein to act so versatile, modulating the antibody response while retaining the ACE2 affinity.
What mutations modify affinity?
SARS-CoV-2 receptor binding domain (RBD) deep mutational scanning approaches identified constraints in the protein’s conformation regarding its ability to interact with ACE2, but also highlighted that many mutations in RBD are well tolerated and do not significantly affect the binding affinity. In fact, many modifications were found to improve binding with ACE2 either by directly increasing affinity, as with mutations N501F, N501T and Q498Y, or by increasing expression of RBD were found for mutations V367F and G502D. Mutations in the ACE2 receptor may also have similar amplifying effects with respect to binding to RBD.
RBD mutations N439 / R426, L452 / K439, T470 / N457, E484 / P470, Q498 / Y484 and N501 / T487 have been noted in particular to increase the binding affinity with ACE2. Several mutant strains of SARS-CoV-2, including the British variant B.1.1.7, the South African variant B.1.351 and the strain P.1 (formerly B.1.1.28.1) identified in Tokyo each exhibit a part or all of these mutations, and are noted for their highest transmissibility.
The mutations that were involved in the leakage of the monoclonal antibodies REG10987 and REG10933 were found to have only a minor negative impact on ACE2 binding and RBD folding. The mutation at the E484 site had the greatest effect on antibody binding, which is in line with other studies that note a ten-fold loss in antibody affinity to RBD due to this change.
The group applied the same structural and functional studies to the antibodies induced by the anti-SARS-CoV-2 mRNA vaccine and to the mutants of importance, noting significantly that the affinity of the antibodies towards the K417N / E484K / N501Y mutants was lower by a small but significant margin. Vaccine-induced immunity may therefore be compromised by other strains of SARS-CoV-2. These key mutations have also been shown to reduce or eliminate the affinity of 14 of the 17 most widely used monoclonal antibodies against the virus.
Crystal structures of the SARS-CoV-2 RBD and S1 domain complexes with ACE enzyme and REGN-COV2 antibody cocktail. (A) Structural overview of the RBD SARS-CoV-2 complex with ACE2 (pdb id 6M0J). The SARS-CoV RBD is shown in cyan ribbons and the RBM region in blue ribbons. Human ACE2 subdomain I is shown in red ribbons and subdomain II is shown in green ribbons. The structure of ACE2 consists of the N-terminal I subdomain (residues 19-102, 290-397 and 417-430) and the C-terminal II subdomain (residues 103-289, 398-416 and 431-615 ) that form the opposite sides of the active site slit. (B) The crystal structure of the S1 domain form dissociated in the complex with ACE2 (pdb id 7A91). S1-RBD is cyan ribbons and ACE2 is green ribbons. (C) The crystal structure of the fully dissociated S1 domain in the complex with ACE2 (pdb id 7A92). The S1 domain of the SARSCoV-2 S protein is cyan ribbons and ACE2 is green ribbons. (D) The cryo-EM structure of the SARS-CoV-2 RBD in the complex with the antibody cocktail REGN10933 / REGN10987. The RBD region is represented by a green surface. The Fab fragment REGN10933 is shown in ribbons with a heavy chain in cyan and a light chain in blue. REGN10987 is ribbons with heavy chain in orange and light chain in purple. The positions of the functional residues targeted by the mutation variants and the mutations escaping the antibodies are E406, K417, E484 and N501 are annotated and highlighted as black spots on the RBD surface. (E) A close-up of SARS-CoV-2 RBD interactions with REGN10933. The RBD is displayed in green area. The Fab fragment REGN10933 is shown in ribbons with a heavy chain in cyan and a light chain in blue. The REGN10933 antibody epitope on RBD is highlighted in cyan patches on the surface. The positions of E406, K417, E484, F486, N501 are represented by black surface spots on the RBD. (F) A close-up of the SARSCoV-2 RBD interface with REGN10987. The red spots correspond to the epitope REGN10987. The positions of E406, K417, N439, E484, F486, N501 are represented by black surface spots on the RBD.
How can ACE2 affinity increase while antibody affinity is decreased?
Residues K417, E484 and N501 have been shown to be key centers of interaction with a high degree of structural plasticity which allow mutants at these positions to improve binding affinity with ACE2. Residues E406, N439, K417, and N501 act as allosteric centers that anchor segments of the molecule together to mediate long-distance interactions. Mutations at these sites alter the structure of the protein and neutralize the ability of monoclonal antibodies to bind to the SARS-CoV-2 spike protein while maintaining ACE2 affinity. These sites are at the hinge centers in the spike protein and are usually involved in the collective movements of the molecule. For example, the RBD is constantly in motion, alternating between closed and open conformations. This explains the ability of these mutations to aid in the escape of antibodies while maintaining or increasing the ACE2 affinity.
Strains of SARS-CoV-2 which are unable to bind to ACE2 are directly evolutionarily disadvantaged and are rapidly eliminated from the population. The highly adaptable and plastic allosteric centers allow the collective movements of the protein to be disrupted, thus becoming immune to monoclonal antibodies that require a specific structural arrangement to interact. The static conformation of the ACE2 interaction face of the spike protein remains largely unaffected by these mutations, only the behavior of the protein arrangement.
*Important Notice
bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behaviors or be treated as established information.
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