Method for analyzing the affinity of a spinous process protein receptor binding domain to a hACE2 receptor molecule and applications thereof

Abstract

The application discloses a method for analyzing the affinity of a spinous process protein receptor binding domain to an hACE2 receptor molecule and an application thereof, and relates to the technical field of biology. The affinity analysis method can avoid the limitations of studying single site mutations and the inability to reflect the interactive influence or synergistic effect of other mutation sites in the local region, and can reasonably divide subdomains, and uniquely design experiments and systematically detect the influence of mutations of each subdomain of the novel coronavirus on the RBD affinity, and compare the influence of single site mutations to verify the key sites and key subdomains of the high affinity of RBD and ACE2.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for analyzing the affinity of the spike protein receptor-binding domain for hACE2 receptor molecules and its application. Background Technology

[0002] The S1 domain of the spike protein includes the N-terminal domain (NTD), receptor-binding domain (RBD), C-terminal domain 1 (CTD1), and C-terminal domain 2 (CTD2). The S1 domain is responsible for the binding of the SARS-CoV-2 virus to the angiotensin-converting enzyme 2 (hACE2) receptor molecule on the surface of human alveolar cells during the initial stage of SARS-CoV-2 infection of the human respiratory tract. After the RBD binds to hACE2, it typically triggers changes in the overall molecular structure and conformation of the spike protein. The RBD / hACE2 complex is cleaved by the transmembrane protease TMPRSS2 on the surface of alveolar cells at the junction of the S1 and S2 domains, causing it to detach and exposing the S2 domain. The fusion peptide (FP) of the S2 domain is released from its original folded structure and inserts into the infected cell membrane. Then, through folding between the FP and the HR1 and HR2 subdomains of the S2 domain, a coiled-coil structure is formed, bringing the viral membrane and the infected cell membrane closer together, promoting membrane fusion and completing the viral infection process.

[0003] Currently, the primary epitope targeted by COVID-19 vaccines and antibodies is the receptor-binding domain (RBD) of the novel coronavirus spike protein. However, the challenge of rapidly and effectively eradicating COVID-19 lies in its spread within the population, its continuous mutation in response to increased human immunity, and its struggle against the development of new vaccines, allowing it to evade the human immune system. Consequently, the development of vaccines and antibodies often fails to effectively eliminate newly discovered circulating strains of the novel coronavirus, posing a severe challenge to COVID-19 prevention and control.

[0004] Therefore, there is a need to provide a method for analyzing the affinity of the spike protein receptor-binding domain for the ACE2 receptor molecule. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for analyzing the affinity of the spike protein receptor-binding domain (RBD) for the ACE2 receptor molecule. This method avoids the limitations of studying single-site mutations and their effects, which cannot reflect the interaction or synergistic effects of other mutation sites in a local region. Through reasonable subdomain division, unique experimental design, and systematic techniques, this invention comprehensively detects the impact of mutations in various subdomains of the SARS-CoV-2 virus on the affinity of the RBD, and compares the effects of single-site mutations to verify the key sites and important subdomains of high affinity between the RBD and ACE2.

[0006] This invention also provides the application of the above affinity analysis method in the design, screening, validation, or antibody development of chemical molecular inhibitors of the novel coronavirus.

[0007] A method for analyzing the affinity of the spike protein receptor-binding domain to the ACE2 receptor molecule according to a first aspect of the present invention includes the following steps:

[0008] S1. By comparing the complex structure formed by spike protein RBD and ACE2 of the prototype SARS-CoV-2 and other closely related viruses, the interaction interface between spike protein RBD and ACE2 is divided into different subdomains with the convex ends and the middle concave region.

[0009] S2. Using the spike protein of the novel coronavirus prototype strain as a template, at least one of the subdomains is replaced with the corresponding subdomain of the spike protein RBD of other closely related viruses to form a domain replacement chimeric protein.

[0010] S3. Using the spike protein RBD protein DNA molecule of the novel coronavirus prototype strain as a backbone, an expression plasmid for replacing the chimeric protein with the aforementioned domain was constructed using molecular biology methods.

[0011] S4. Expression and purification yield the domain-replaced chimeric protein, which is the chimeric mutant protein of the SARS-CoV-2 spike protein.

[0012] S5. The binding kinetic parameters of the chimeric mutant protein of the SARS-CoV-2 spike protein are measured by SPR technology, and the binding kinetic parameters of the spike protein of the SARS-CoV-2 prototype are referenced to analyze the influence of each subdomain on the interaction between the spike protein RBD and ACE2.

[0013] S6. Based on the analysis in step S5, the effects of different subdomains of the novel coronavirus spike protein on the RBD / ACE2 interaction were confirmed, and the key domain of the novel coronavirus spike protein RBD acting on the ACE2 receptor molecule with high affinity was identified. Single-point mutations were performed on key amino acid residues in the key domain of the prototype strain of the novel coronavirus spike protein to obtain the single-point mutant protein of the novel coronavirus spike protein RBD.

[0014] S7. The binding kinetic parameters of the SARS-CoV-2 spike protein RBD single-point mutant protein are measured by SPR technology, and the binding kinetic parameters of the spike protein of the SARS-CoV-2 prototype strain are referenced to analyze the influence of subdomains and single-point mutations on intermolecular interactions.

[0015] S8. Combining the analytical results of steps S1 to S7 with the molecular interaction mechanism of spike protein RBD and ACE2, the high-affinity interaction subdomain on the interface of the RBD and ACE2 complex was confirmed.

[0016] The term "other closely related viruses" refers to coronaviruses belonging to the same subgenus *sarbecovirus* as the prototype strain of SARS-CoV-2. For example, with SARS-CoV-2 as the prototype strain of SARS-CoV-2, other closely related viruses could be Omicron B.1.1529, SARS-CoV-1, RaTG13, or Omicron BA5.2.

[0017] The affinity analysis method according to embodiments of the present invention has at least the following beneficial effects:

[0018] The SARS-CoV-2 spike protein RBD is the protein-binding domain that binds to hACE2. The RBD protein domain encodes 223 amino acid residues. At the interface with hACE2, its structure is characterized by two ridge-like protrusions at each end and a concave region in the middle. The N-terminal α1 helix of the hACE2 protein is the main part contacting the RBD, spanning both ends, while the middle portion resides on the concave substructure in the middle of the RBD. Therefore, characterizing the two molecules, the N-terminal α1 helix of hACE2 rides on the saddle-like interface of the RBD. Studies of the protein complex structure reveal the existence of locally dense networks of polar or hydrophobic interactions throughout the helical contact region.

[0019] Existing research on the affinity mechanism between RBD and hACE2 is generally limited to describing the polar or nonpolar interactions between specific amino acid residues in the structure of a particular RBD / hACE2 protein complex, failing to reveal the fine-grained behavior of these molecules. The affinity analysis method described in this invention avoids the limitations of studying single-site mutations and their effects, which cannot reflect the interactions or synergistic effects of other mutation sites in a local region. Through reasonable subdomain division, unique experimental design, and systematic techniques, it comprehensively examines the impact of mutations in each subdomain of the SARS-CoV-2 virus on RBD affinity. It also employs precise quantitative characterization of the impact of mutational changes on protein-protein interactions. Surface plasmon resonance (SPR) technology is used to accurately measure the protein-protein interaction between the SARS-CoV-2 RBD protein and its mutant proteins and ACE2. The dynamic equilibrium dissociation constant and changes in protein-protein affinity are analyzed from the kinetic measurements of RBD and ACE2 binding and dissociation, comparing the effects with single-site mutations to verify the key sites and important subdomains of high affinity between RBD and ACE2. Furthermore, the contribution of each subdomain can be quantitatively characterized. This affinity analysis method provides an important reference for the design and development of related SARS-CoV-2 inhibitory drugs, highly effective vaccines, and antibodies.

[0020] This invention focuses on dividing the interface between the spike protein RBD and ACE2 into three subdomains. In traditional research models, the concave region and a raised crest are typically grouped into one region, but the local interaction patterns of the amino acid residues in these two merged regions are not identical. Furthermore, based on protein molecular dynamics simulations, computational simulations show that RBD binds to ACE2 in a dynamic binding pattern that sequentially closes from the concave region to the aforementioned raised crest. This indicates that different molecular subdomains on the RBD / ACE2 interface influence the way the two molecules interact, and the nature of these interactions may differ. Given that the affinity between RBD and ACE2 varies in the β-coronavirus family, especially SARS-CoV-2, despite the emergence of mutation hotspots, there remains a high degree of consistency among different RBD / ACE2 protein complex structures, this invention, by replacing these subdomains and combining them with SPR technology, can accurately assess the impact of specific local interaction networks of these regions on intermolecular interactions, including their effects on molecular dynamics, based on currently published protein structures.

[0021] Furthermore, the research samples of this invention include not only novel coronavirus mutant strains but also viral strains on the phylogenetic tree that are very closely related to SARS-CoV-2 but have drastically different affinities. By employing spatial structure partitioning to compare the binding kinetics of the spike proteins of β-coronaviruses and prevalent novel coronavirus mutant strains with different affinities to ACE2, this approach can reveal, in a broader sense, the molecular mechanism by which coordination between subdomains promotes the interaction between RBD and ACE2.

[0022] According to some embodiments of the present invention, the ACE2 protein includes, but is not limited to, ACE2 proteins of humans, monkeys, mice, cats, dogs, pigs, white-tailed deer, chickens, ducks, and horses.

[0023] According to some embodiments of the present invention, by analyzing the molecular structure of the novel coronavirus spike protein RBD / ACE2 complex, the differences in key points and spatial distribution of the interaction contact region between the spike protein and ACE2 are mainly considered to identify the subdomains in which the RBD contacts the N-terminal α1 helix of the receptor molecule ACE2. Specifically, this includes the CR1 subdomain in contact with the N-terminus of the ACE2 N-terminus α1 helix, the CR2 subdomain with a central indentation, and the CR3 subdomain in contact with the C-terminus of the ACE2 N-terminus α1 helix at the other end.

[0024] Due to the high homology between the coronavirus spike protein RBD and the ACE2 complex, the division of specific subdomains is based on the crystal structure of the prototype novel coronavirus RBD / hACE2 protein complex (pdb#6M0J). The spatial boundaries of adjacent CR1 and CR2 are based on the spatial distribution of amino acid residues Y449 / G496, and the spatial boundaries of adjacent CR2 are based on the spatial distribution of amino acid residues F456 / F490.

[0025] According to some embodiments of the present invention, in step S2, replacing the subdomain with the corresponding subdomain of other closely related viral spike protein RBD specifically involves: comparing the homologous sequences and homologous structures of the prototype SARS-CoV-2 and other closely related viral spike protein RBD sequences, and selecting the contact surfaces of each RBD / ACE2 structural complex adjacent to each other. Amino acid residues within a certain distance serve as important discrimination sites for interactions. Based on the differences in the corresponding amino acid residues in the subdomains of the spike protein RBD of the prototype SARS-CoV-2 and other closely related viruses, the amino acids at the important discrimination sites for interactions in the subdomains of the spike protein RBD of the prototype SARS-CoV-2 are replaced with the corresponding amino acids in the spike protein RBD of other closely related viruses through single-point mutation or multi-point mutation.

[0026] According to some embodiments of the present invention, in step S1, dividing the interaction surface between the spike protein RBD and ACE2 into different subdomains based on the protruding ends and the concave region in the middle specifically involves dividing the interaction between the spike protein RBD and the ACE2 molecule according to its morphology and region. Specifically, the spike protein RBD is divided into three subdomains. More specifically, the spike protein RBD is divided into a ridge-like subdomain (CR1 / CR3) protruding at both ends and a concave subdomain (CR2) in the middle. The CR1 subdomain consists of I472-490F; the CR2 subdomain consists of K417 and Y449-F456, F490-G496; and the CR3 subdomain consists of G496-505Y, K444-Y449.

[0027] According to some embodiments of the present invention, the other closely related viruses include at least one of the following: Omeprone mutant strain BA.1.1.529, Omeprone mutant strain BA.5.2, RaTG13 coronavirus, and SARS-CoV-1.

[0028] According to some embodiments of the present invention, in step S3, constructing the expression plasmid of the domain-replacing chimeric protein using molecular biology methods specifically includes: replacing the amino acid-coding nucleotides of the interaction-critical discrimination sites in the RBD subdomain of the prototype SARS-CoV-2 spike protein with the corresponding amino acid-coding nucleotides of the RBD of other closely related viral spike proteins using molecular biology methods. This can be achieved through single-point mutation or multi-point mutation.

[0029] According to some embodiments of the present invention, in step S4, the expression domain replacement chimeric protein includes: culturing cells containing the corresponding expression vector. Transient in vitro expression can be performed using cell engineering methods.

[0030] According to some embodiments of the present invention, in step S4, the purified domain-replacement chimeric protein can be purified by any protein purification method known in the art.

[0031] According to some embodiments of the present invention, the purity of the SARS-CoV-2 spike protein chimeric mutant protein is 90% or higher.

[0032] According to some embodiments of the present invention, the N-terminus and / or C-terminus of the SARS-CoV-2 spike protein chimeric mutant protein are tagged.

[0033] According to some embodiments of the present invention, the N-terminus and / or C-terminus of the SARS-CoV-2 spike protein RBD single-point mutant protein are tagged.

[0034] According to some embodiments of the present invention, the tag includes at least one of the tags that facilitate the dissolution, transfer, purification and detection of the chimeric mutant protein of the SARS-CoV-2 spike protein or the single-point mutant protein of the SARS-CoV-2 spike protein RBD. For example: tags that facilitate the dissolution of the chimeric mutant protein of the SARS-CoV-2 spike protein or the single-point mutant protein of the SARS-CoV-2 spike protein RBD include, but are not limited to, nus tags or maltose-binding proteins; tags that facilitate the transfer of the chimeric mutant protein of the SARS-CoV-2 spike protein or the single-point mutant protein of the SARS-CoV-2 spike protein RBD include, but are not limited to, the signal peptide sequence of the interleukin-2 (IL-2) protein; tags that facilitate the purification of the chimeric mutant protein of the SARS-CoV-2 spike protein or the single-point mutant protein of the SARS-CoV-2 spike protein RBD include, but are not limited to, strep tags, His tags, GST tags, pelB signal sequences, or ompA signal sequences; tags that facilitate the detection of the chimeric mutant protein of the SARS-CoV-2 spike protein or the single-point mutant protein of the SARS-CoV-2 spike protein RBD include, but are not limited to, horseradish peroxidase (HRP), β-galactosidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, or cyan fluorescent protein (CFP). Specifically, the tag can be a strep tag.

[0035] It is understood that the SARS-CoV-2 spike protein chimeric mutant protein or the SARS-CoV-2 spike protein RBD single-point mutant protein of the present invention may contain one or more tags; multiple tags may include a combination of multiple identical tags or a combination of multiple different tags.

[0036] According to some embodiments of the present invention, in step S5, analyzing the effect of each subdomain on the interaction between the spike protein RBD and ACE2 includes: comparing the affinity of the prototype SARS-CoV-2 spike protein and the SARS-CoV-2 spike protein chimeric mutant protein, and identifying the contribution of the subdomain to the affinity of RBD and ACE2. The comparison includes comparisons between the same subdomain and comparisons between different subdomains.

[0037] According to some embodiments of the present invention, in step S5, the effects of different subdomains of the selected Omeprone mutant strains BA.1.1.529, BA.5.2, RaTG13 coronavirus, and SARS-CoV-1 on the molecular interaction between the spike protein of the prototype SARS-CoV-2 and hACE2 after substitution on the spike protein of the prototype SARS-CoV-2 are comprehensively considered, and the key molecular subdomain affecting the interaction between the RBD domain of the spike protein of SARS-CoV-2 and hACE2 is determined to be the CR3 subdomain.

[0038] According to some embodiments of the present invention, the combined kinetic parameters include the dissociation equilibrium kinetic parameter K. D .

[0039] According to some embodiments of the present invention, step S6 obtains the SARS-CoV-2 spike protein RBD single-point mutant protein, including expressing and purifying the SARS-CoV-2 spike protein RBD single-point mutant protein.

[0040] According to some embodiments of the present invention, the purity of the SARS-CoV-2 spike protein RBD single-point mutant protein is 90% or higher.

[0041] According to some embodiments of the present invention, step S8 further includes: accurately resolving the contribution of the high-affinity interaction subdomain to the high-affinity interaction between molecules based on the crystallographic structure and cryo-electron microscopy structure of the spike protein RBD and ACE2 complex of the prototype strain of SARS-CoV-2 and other closely related viruses, and clarifying the molecular mechanism and intrinsic laws.

[0042] According to some embodiments of the present invention, based on the crystallographic and cryo-electron microscopy structures of the reported prototype strain SARS-CoV-2, Omeprone mutant strains (BA.1.1.529, BA.5.2), RaTG13 coronavirus, SARS-CoV-1 spike protein RBD and hACE2 complex, the contribution of the CR3 subdomain to the high affinity intermolecular forces is precisely resolved, and the molecular mechanism and intrinsic laws are clarified.

[0043] According to some embodiments of the present invention, the high affinity mechanism of CR3 includes: firstly, the formation of local hydrogen bond, electrostatic interaction, or hydrophobic interaction networks in the subdomain; secondly, for the local inter-residue interaction networks, the screened high affinity contributing local interaction networks exhibit the following characteristics: ① In the RBD CR3 subdomain of the identified SARS-CoV-2 Omicryn mutant strain, there are two local interaction networks: taking BA.5.2 as an example, hACE2's Y41 and RBD's T500 / Y501, hACE2's Q42 / D38 and R498 / Y449 / respectively form two spatially relatively dense inter-residue interaction networks, with the interactions within the networks mainly consisting of hydrogen bonds and π-π hydrophobic interactions; ② The reason for the high affinity of the Omicryn mutant strain CR3 is that each locally dense molecular interaction network forms two relatively consistent pulling forces, fixing the molecular binding of RBD CR3 and hACE2. ③ When two local interaction networks are linked together, the combined attraction can generate a stronger intermolecular attraction. This attraction can be a π-π hydrophobic interaction between the two local interaction networks, or a combined hydrogen bond interaction. This feature is particularly prominent in the CR3 region of SARS-CoV-1, which is also the reason why its CR3 chimeric protein generates super affinity.

[0044] According to some embodiments of the present invention, in step S7, the selected sites are Y505, N501, G496, G446, G496, and Q498R, which are mutated to the corresponding amino acid residues at the mutation sites in the prevalent Omicrong mutant strains (BA.1.1.529 and BA.5.2). Detection revealed that the N501Y mutation significantly enhanced the affinity for hACE2 compared to the original SARS-CoV-2 RBD (5-fold increase), and the Q498Y mutation also significantly enhanced intermolecular forces (2-fold increase compared to the original RBD), while the G496S / Y505 mutation significantly reduced intermolecular interactions (5-fold and 8-fold weaker than the original RBD, respectively). This provides strong evidence for verifying the molecular mechanism for generating the super-affinity of CR3 proposed above, especially the enhanced merging of the pull between the two local interaction networks inherent in the super-affinity action of CR3 in SARS-CoV-1. Furthermore, the significant local inhibition of Y505H, compared with the strong affinity of the overall CR3 subdomain of the Omicrong mutants (BA.1.1.529 and BA.5.2) containing this mutation (the corresponding subdomain replacement mutants showed an affinity for hACE2 that was more than 20 times higher than the original RBD), corroborates that the effect of single-point amino acid mutations in the domain should be interpreted from the perspective of the overall domain, rather than being limited to single-point considerations. This demonstrates the effectiveness of analyzing molecular effects by partitioning the spike protein subdomains, objectively reflects the importance of focusing on key domains in molecular mechanisms of action, and provides a new perspective and focus for studying the affinity between the novel coronavirus spike protein and ACE2.

[0045] The above-described affinity analysis method according to the second aspect of the present invention is applied in the design, screening, validation, or antibody development of chemical molecular inhibitors of the novel coronavirus.

[0046] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the contact surface of the complex formed by the RBD of the spike protein and hACE2; Figure A is a schematic diagram of the partitioned structure of the contact surface of the complex, and Figure B is a schematic diagram of the RBM part of the RBD protein domain in contact with hACE2 divided into three consecutive subdomains (CR1 / 2 / 3) based on the crystal diffraction molecular structure of the complex formed by the RBD of the spike protein of the prototype SARS-CoV-2 spike protein and hACE2 (pdb#6M0J) and the local interaction network formed between important amino acid residues.

[0048] Figure 2Figure 1 shows the contact residue analysis results between CR1, CR2, and CR3 of the spike protein and hACE2; Figure A shows the alignment results of the template structure and homologous sequences, with contact amino acid residues located at the interface between the spike protein RBD and hACE2 marked with black arrows; Figure B shows the results based on... Figure 1 The partitioning of the RBD subdomain of the prototype SARS-CoV-2 virus and the crystal structure of the RBD / hACE2 protein complex (pdb#, 6M0J) bring the two molecules together at the interface. Amino acid residues within the distance are used as direct interaction reference points. Residues corresponding to the RBD of other coronavirus strains and SARS-CoV-2 derived virus strains are statistically analyzed and listed, and residues with mutation points are selected and marked in red.

[0049] Figure 3 The results are shown in the SDS-PAGE electrophoresis of each chimeric mutant protein; lane 1 is the protein marker, and lanes 2, 3, 4, and 5 are for gradient elution and sample collection in separate tubes.

[0050] Figure 4 The effect of subdomain substitution on the affinity of SARS-Cov-2 RBD / hACE2 interaction;

[0051] Figure 5 The results of SDS-PAGE electrophoresis of single-point mutant proteins of the spike protein RBD of the SARS-CoV-2 virus are shown. Lane 1 is the protein marker, and lanes 2, 3, and 4 are for samples collected by gradient continuous elution.

[0052] Figure 6 The effects of key point mutations in the CR3 subdomains Y505H, N501Y, G496S, G446S, and Q498R on the affinity of the RBD domain of the prototype SARS-CoV-2 spike protein for hACE2.

[0053] Figure 7Figure 1 shows the crystal structures of complexes formed by the CR3 subdomain of the RBD and hACE2 of different SARS-CoV-2 spike protein strains. Figure A shows the molecular structure of the local interaction between the CR3 subdomain of the SARS-CoV-1 spike protein RBD and hACE2, with the selected molecular structure being pdb#2AJF; Figure B shows the molecular structure of the local interaction between the CR3 subdomain of the PT SARS-CoV-2 spike protein RBD and hACE2, with the selected molecular structure being pdb#6M0J; Figure C shows the molecular structure of the local interaction between the CR3 subdomain of the SARS-CoV-2 omicron (BA.1.1.529) spike protein RBD and hACE2, with the selected molecular structure being pdb#7TN0; Figure D shows the molecular structure of the local interaction between the CR3 subdomain of the SARS-CoV-2 omicron (BA.5.2) spike protein RBD and hACE2, with the selected molecular structure being pdb#7XWA; Figure E shows the crystal structure of the Bat RaTG3 spike protein RBD. The molecular structure of the local interaction between the CR3 subdomain and hACE2 is shown in Figure F, which is pdb#7DRV. Figure F shows the molecular structure of the local interaction between the CR3 subdomain of the SARS-Cov-1 spike protein RBD and the corresponding CR3 region of the specific high-affinity antibody 80R, which is pdb#2GHW. The hydrogen bonds or ionic electrostatic interactions between amino acid residues are represented by yellow dashed lines. The hydrophobic π-π interaction between the benzene ring of Y41 of hACE2 and the hydroxybenzene rings of Y484 (SARS-Cov-1) / Y501 (SARS-Cov-2) of RBD is represented by purple dashed lines, and the distance between the centers of the benzene rings is marked. Detailed Implementation

[0054] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0055] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0057] Example 1

[0058] A method for analyzing the affinity of the spike protein receptor-binding domain for the hACE2 receptor molecule, comprising the following steps:

[0059] S1. Based on the reported crystal diffraction molecular structure of the complex formed by the RBD and hACE2 of the prototype SARS-CoV-2 spike protein and the local interaction network formed between important amino acid residues, and compared with the crystal diffraction molecular structure of the complex formed by the RBD and hACE2 of other closely related viral spike proteins, the receptor binding motif (RBM) part of the RBD and hACE2 contact is divided into three spatially continuous subdomains (CR1, CR2 and CR3).

[0060] Specifically, the prototype strain of SARS-CoV-2 has a crystal diffraction molecular structure of the complex formed by the RBD of its spike protein and hACE2, which is pdb#6M0J. It is divided into ridge-like subdomains (CR1 / CR3) protruding at both ends of the RBD and a recessed subdomain (CR2) in the middle. The CR1 subdomain of the spike protein of the prototype strain of SARS-CoV-2 is I472-490F, which is the ridge-like subdomain protruding at one end that binds to hACE2. The CR2 subdomain is composed of K417 (this site is directly involved in the contact between the recessed region and hACE2 and plays a key role) and Y449-F456, F490-G496, which is the recessed subdomain located in the middle part of the RBD. The CR3 subdomain is composed of G496-505Y, K444-Y449. The two amino acid residues are not continuous in sequence, but together in spatial structure they form the ridge-like subdomain protruding at the other end that binds to hACE2.

[0061] Based on the intermolecular contact regions and the distribution of key amino acids, the adjacent boundaries of CR1 and CR2, and CR2 and CR3, are divided according to the positions of F456 / F490 and Y449 / G496, respectively. For example... Figure 1 As shown.

[0062] Other closely related viruses are Omicron B.1.1 529, SARS-CoV-1, RaTG13, and SARS-CoV-2 Omicron BA5.2, with corresponding complex crystal diffraction molecular structures of pdb#7TN0, pdb#2AJF, pdb#7DRV, and pdb#7XWA. The spike protein RBD of other closely related viruses shows high structural similarity and sequence homology with the prototype SARS-CoV-2 virus, and the division of the CR1, CR2, and CR3 subdomains is the same as that of the prototype SARS-CoV-2 virus.

[0063] S2. Using the RBD sequence and structure of the spike protein of the SARS-CoV-2 prototype strain as a template, obtain the contact residues of CR1, CR2 and CR3 of the spike protein of the prototype strain of SARS-CoV-2 and other closely related viruses with hACE2, and confirm the mutation sites in the contact residues of other closely related viruses.

[0064] Based on the subdomains of the prototype SARS-CoV-2 spike protein RBD and the above analysis results, the mutation sites of each subdomain (CR1, CR2 and CR3) were replaced accordingly to obtain 12 chimeric mutants, and the corresponding protein sequences and gene sequences were obtained.

[0065] Specifically, the contact residues between CR1 and hACE2 of the spike protein of the novel coronavirus prototype strain SARS-CoV-2 include A475, F486, N487, and Y489; the contact residues between CR2 and hACE2 include K417, Y453, L455, F456, and Q493; and the contact residues between CR3 and hACE2 include G446, Y449, G496, Q498, T500, N501, G502, and Y505.

[0066] For other closely related viral spike proteins (including spike proteins from other selected coronaviruses or spike proteins from SARS-CoV-2-derived mutant strains), the alignment of their template structures and homologous sequences (e.g.) is crucial. Figure 2 As shown in Figure A, the contact residues and mutation information of CR1, CR2, and CR3 with hACE2 were obtained (e.g., Figure 2 (As shown in Figure B). Details are as follows:

[0067] ① The corresponding mutation sites on the RBD of the spike protein of SARS-CoV-2 Omicron (B.1.1.529) were identified. The mutation sites of CR1 (CR1-Omicron (B.1.1.529)) are S477N, T478K, and E484A; the mutation sites of CR2 (CR2-Omicron (B.1.1.529)) are K417N and Q493R; and the mutation sites of CR3 (CR3-Omicron (B.1.1.529)) are G446S, G496S, Q498R, N501Y, and Y505H.

[0068] ② The mutation sites on the spike protein RBD of SARS-CoV-1 were determined. The mutation sites of CR1 (CR2-SARS) are A475P and F486L; the mutation sites of CR2 (CR2-SARS) are K417V, L455Y, F456L, and Q493N; and the mutation sites of CR3 (CR2-SARS) are G446T, Q498Y, and N501T.

[0069] ③ The mutation sites on the RaTG13 spike protein RBD were determined. The mutation site of CR1 (CR3-RaTG13) is F486L; the mutation site of CR2 (CR2-RaTG13) is Q493Y; and the mutation sites of CR3 (CR3-RaTG13) are Y449F, Q498Y, N501D, and Y505H.

[0070] ④ The corresponding mutation sites on the RBD of the SARS-CoV-2 Omicron (BA5.2) spike protein were determined. The mutation sites of CR1 (CR1-Omicron((BA5.2)) are S477N, T478K, E484A, F486V; the mutation sites of CR2 (CR2-Omicron((BA5.2)) are K417N, L452R; and the mutation sites of CR3 (CR3-Omicron((BA5.2)) are Q498R, N501Y, Y505H.

[0071] Specifically, ① CR1, CR2, and CR3 of SARS-CoV-2 Omicron (BA.5.2) were replaced with the corresponding subdomains of the spike protein of the prototype SARS-CoV-2 strain, respectively, to obtain SARS-CoV-2RBD-CR1-Omicron (BA.5.2), SARS-CoV-2RBD-CR3-Omicron (BA.5.2), and SARS-CoV-2RBD-CR3-Omicron (BA.5.2), with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively;

[0072] ② Replace CR1, CR2, and CR3 of SARS-CoV-2 Omicron ((B.1.1.529) with the corresponding subdomains of the spike protein of the prototype SARS-CoV-2 strain, respectively, to obtain SARS-CoV-2RBD-CR1-Omicron (B.1.1.529), SARS-CoV-2RBD-CR2-Omicron (B.1.1.529), and SARS-CoV-2RBD-CR3-Omicron (B.1.1.529), with amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively;

[0073] ③ Replace CR1, CR2, and CR3 of the RaTG13 virus strain with the corresponding subdomains of the spike protein of the prototype SARS-CoV-2 strain, respectively, to obtain SARS-CoV-2RBD-CR1-RaTG13, SARS-CoV-2RBD-CR2-RaTG13, and SARS-CoV-2RBD-CR3-RaTG13, respectively, with amino acid sequences as shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively;

[0074] ④ Replace CR1, CR2 and CR3 of SARS-CoV-1 with the corresponding subdomains of the spike protein of the prototype SARS-CoV-2, respectively, to obtain SARS-CoV-2RBD-CR1-SARS, SARS-CoV-2RBD-CR2-SARS and SARS-CoV-2RBD-CR3-SARS, respectively, with amino acid sequences as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

[0075] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.1);

[0076] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.2);

[0077] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.3);

[0078] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.4);

[0079] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLRSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.5);

[0080] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.6);

[0081] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGLNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.7);

[0082] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLYSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.8);

[0083] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNFNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFYPTDGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.9);

[0084] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQPGSTPCNGVEGLNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.10);

[0085] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRYLRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLNSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.11);

[0086] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVTGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFYPTTGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(SEQ ID NO.12).

[0087] S3. Obtain gene fragments encoding the spike protein RBD of the prototype SARS-CoV-2 virus and expression vectors of chimeric mutants obtained in step S3.

[0088] The specific steps are as follows:

[0089] S31. Using molecular biology methods, the gene fragment encoding the spike protein RBD of the prototype SARS-CoV-2 strain was cloned into an expression vector. A signal peptide sequence was fused to the 5' end of the gene fragment to guide the protein's secretory expression in mammalian cells. A 6×His tag was fused to the 3' end of the gene fragment to construct the prototype SARS-CoV-2 spike protein RBD expression vector (pCDNA3.1-IL2 SP-SARS2-RBD-6×His).

[0090] The expression vector was pcRNA3.1; the signal peptide sequence was the signal peptide sequence of interleukin-2 (IL-2) protein.

[0091] The gene fragment encoding the spike protein RBD of the prototype SARS-CoV-2 virus is shown in SEQ ID NO.13 (GenBank:NC_045512.2);

[0092] (SEQ ID NO.13).

[0093] S32. Based on the vector pCDNA3.1-IL2 SP-SARS2-RBD-6×His, a chimeric mutant was constructed by replacing a specific subdomain of the prototype SARS-CoV-2 spike protein RBD using point mutation PCR or seamless cloning.

[0094] The gene fragments encoding the spike protein of the prototype SARS-CoV-2, namely CR1, CR2, and CR3, were replaced with the corresponding fragments in SARS-CoV-2Omicron (B.1.1.529), respectively, to obtain the vectors pCDNA3.1-IL2 SP-{SARS2-RBD-CR1-Omicron(B.1.1.529)}-6×His, pCDNA3.1-IL2 SP-{SARS2-RBD-CR2-Omicron(B.1.1.529)}-6×His, and pCDNA3.1-IL2 SP-{SARS2-RBD-CR3-Omicron(B.1.1.529)}-6×His;

[0095] The gene fragments encoding the spike protein of the prototype SARS-CoV-2, namely CR1, CR2, and CR3, were replaced with the corresponding fragments in SARS-CoV-2Omicron(BA.5.2), respectively, to obtain the vectors pCDNA3.1-IL2 SP-{SARS2-RBD-CR1-Omicron(BA.5.2)}-6×His, pCDNA3.1-IL2 SP-{SARS2-RBD-CR2-Omicron(BA.5.2)}-6×His, and pCDNA3.1-IL2 SP-{SARS2-RBD-CR3-Omicron(BA.5.2)}-6×His;

[0096] The gene fragments encoding the spike protein of the prototype SARS-CoV-2, namely CR1, CR2, and CR3, were replaced with the corresponding fragments from the RaTG13 viral strain, respectively, to obtain the vectors pCDNA3.1-IL2 SP-(SARS2-RBD-CR1-RaTG13)-6×His, pCDNA3.1-IL2 SP-(SARS2-RBD-CR2-RaTG13)-6×His, and pCDNA3.1-IL2 SP-(SARS2-RBD-CR3-RaTG13)-6×His;

[0097] The gene fragments encoding the spike protein of the prototype SARS-CoV-2, namely CR1, CR2, and CR3, were replaced with the corresponding fragments from SARS-CoV-1, resulting in the vectors pCDNA3.1-IL2 SP-(SARS2-RBD-CR1-SARS)-6×His, pCDNA3.1-IL2 SP-(SARS2-RBD-CR2-SARS)-6×His, and pCDNA3.1-IL2 SP-(SARS2-RBD-CR3-SARS)-6×His, respectively.

[0098] The primers used are shown in Table 1.

[0099] Table 1

[0100]

[0101]

[0102] S4. Expression and purification of chimeric mutant protein with RBD subdomain replacement of SARS-CoV-2 spike protein.

[0103] The specific steps are as follows:

[0104] S41. Using PEI 40K transfection reagent (Servicebio), the vector obtained in step S3 was transfected into 293F cells. The cells were cultured at 8% CO2, 37°C, and 130 rpm. After 72 hours of transfection, the cells were centrifuged and the culture supernatant was collected.

[0105] S42. After centrifuging the supernatant at 4℃ and 6000g for 15min, filter the supernatant through a 0.22μm PVDF membrane to obtain the filtrate.

[0106] S43. Affinity purification of the filtrate was performed using a HisTrap HP 5 mL pre-packed column. The column was first equilibrated and washed with solution A (20 mM Tris, 200 mM NaCl, 20 mM Imidazole, pH 7.5); then, proteins were eluted using a gradient of solution B (20 mM Tris, 200 mM NaCl, 500 mM Imidazole, pH 7.5); the elution peaks were concentrated using a 10 kDa MWCO Amicon Ultra 15 mL centrifugal filter (Milipore); gel filtration chromatography was performed using Superdex 200 Increase 10 / 300GL (Cytiva) with buffer solution C (20 mM PBS, 140 mM NaCl, pH 7.4), aliquoted at approximately 200 μL / tube, and stored at -80°C.

[0107] S44. After gel filtration chromatography elution, the purified protein was obtained. The protein concentration was determined using the BCA protein assay kit, and the protein purity was identified by polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining.

[0108] SDS-PAGE test results are as follows: Figure 3 As shown.

[0109] S5. The interaction between the chimeric mutant protein with RBD subdomain substitution of the SARS-CoV-2 spike protein and hACE2 was detected by surface plasmon resonance (SPR) technique, and the effect of subdomain substitution on the affinity of the two molecules was compared.

[0110] The kinetic constants of the interaction between RBD and hACE2 were determined using a BIAcore 8K (GE Healthcare). At 25°C, a multi-cycle capture method was used to capture approximately 500 response values ​​(RU) of hACE2 (1-740)-Fc on a Series S Sensor Chip Protein A chip. The mutant protein (0, 3.7, 11.1, 33.3, 100, 300, 900, 33.3 nM) in the RBD region of the SARS-CoV-2 spike protein was passed through at a flow rate of 30 L / min for 240 seconds and dissociated for 600 seconds. The kinetic parameters were calculated using a 1:1 binding model fitted in BiacoreEvaluation Software (GE Healthcare). The experiment was repeated at least twice.

[0111] The results are as follows Figure 4 As shown in Table 2.

[0112] ① After replacing the CR3 subdomain of the spike protein of RaTG13 with the corresponding subdomain of the RBD of the prototype SARS-CoV-2 spike protein, the RBD chimeric mutant protein did not show an SPR signal for binding to hACE2, indicating that the mutant lost its ability to bind to hACE2. In contrast, after replacing the CR3 subdomain of SARS-CoV-1 and the Omicryn mutants (B.1.1.529 and BA.5.2), these three different chimeric mutants showed a very significant affinity enhancement effect relative to the prototype SARS-CoV-2 RBD (K, K ... D =0.38±0.05nM, K D =1.85±0.12nM and K D =1.03±0.01nM, the affinity of the prototype strain is K. D =44±2.06nM).

[0113] ② After the CR2 subdomain of the RaTG13 spike protein was replaced with the corresponding RBD subdomain of the prototype SARS-CoV-2 spike protein, the RBD chimeric mutant protein showed a significantly enhanced affinity for hACE2 compared to the prototype SARS-CoV-2 RBD (K D =8.8±0.2nM); In contrast, after replacing the CR2 subdomain of SARS-CoV-1, the Omicryn mutant strain (B.1.1.529 and BA.5.2), the affinity of these three different chimeric mutants for the RBD of the prototype SARS-CoV-2 strain was significantly reduced (K = 8.8±0.2nM);D =364±59.4nM, K D =222±52.27nM and K D =179±4.7nM, and the affinity of the prototype strain is K. D =44±2.06nM).

[0114] ③ After the CR1 subdomain of the RaTG13 spike protein was replaced with the corresponding RBD subdomain of the prototype SARS-CoV-2 spike protein, the affinity of the RBD chimeric mutant protein for hACE2 was significantly weakened compared to that of the prototype SARS-CoV-2 RBD (K D =300.67±11.02nM). Unlike the CR3 and CR2 subdomains mentioned above, in the CR1 subdomain, after replacing the CR1 subdomain of the Omicrong mutant strains (B.1.1.529 and BA.5.2), the chimeric mutants containing the Omicrong B.1.1.529 CR1 subdomain showed increased affinity for the RBD of the prototype SARS-CoV-2 virus (K). D =13.59±0.23nM, and the affinity of the prototype strain is K. D =44±2.06nM); while replacing the CR1 subdomain in the Omicrong mutant strain (BA.5.2) resulted in a slight decrease in affinity of chimeras containing this subdomain compared to the prototype SARS-CoV-2 RBD (K0). D =55.26±0.34nM).

[0115] Table 2

[0116]

[0117]

[0118] Note: Replacing the SARS-Cov-1 CR1 chimeric mutant did not yield the protein, so the entire hACE2 contact domain RBM (including CR1 / CR2 / CR3) was replaced as a comparison to detect the coordination relationship between CR1, CR2, and CR3.

[0119] Due to the impact of mutations on protein stability, no chimeric mutant protein of CR1 after substitution by SARS-CoV-1 was obtained. However, considering that the CR1 region of this SARS coronavirus spike protein itself has only two amino acid residue mutations that differ from the prototype SARS-CoV-2 strain, namely F486L and A475P, the former corresponds to the substitution of the CR1 subdomain of the spike protein of RaTG13 (only the F486L mutation), while the latter showed a slight decrease in RBD affinity compared to the prototype SARS-CoV-2 strain in single mutant mutation tests (K). D=52.7±7.2nM), combining the effects of the two mutations, the chimeric mutant containing the CR1 subdomain of the SARS virus spike protein should also have a weakened affinity for hACE2 compared to the prototype SARS-CoV-2 RBD.

[0120] The above results indicate that replacing the CR3 subdomain has a clear and significant impact on the spike protein RBD and hACE2, whether it enhances or weakens them, manifesting as a change in affinity for RBD and hACE2 by tens of times or almost eliminating their affinity activity. CR2 and CR1, however, exhibit different effects, even among the closely related SARS-CoV-2 Omikron mutants (B.1.1.529 and BA.5.2), where the effects of the CR1 and CR2 subdomains are inconsistent. Furthermore, from the perspective of quantifying enhancement or weakening, the influence of CR3 is the most significant.

[0121] This demonstrates that the subdomain partitioning combined with SPR precision measurement technology employed in this invention not only reveals the varying effects of different subdomains of the same coronavirus spike protein RBD on its affinity for hACE2, but also confirms the crucial role of the CR3 subdomain in the interaction between the two molecules, making it the main high-affinity contributing domain. Given the rigor of the research, further investigation will be conducted to identify the influence of key amino acid residues within this subdomain on RBD affinity.

[0122] S6. Based on the subdomain that contributes the main high affinity in the prototype strain SARS-CoV-2 spike protein RBD obtained from step S5, single-point mutations are performed on the amino acid residues that constitute the main polar interaction network of this subdomain, and an expression vector for the SARS-CoV-2 spike protein RBD single-point mutant protein is constructed. The SARS-CoV-2 spike protein RBD single-point mutant protein is then expressed and purified.

[0123] The main polar interaction network of the CR3 subdomain of the spike protein RBD of the prototype SARS-CoV-2 strain is composed of several key amino acid residues: Y505, N501, G496, G446, and Q498. These important amino acid residues were selected for single-point mutation, which were mutated to the corresponding amino acid residues in the popular omicron mutant strains (BA.1.1.529 and BA.5.2). The mutation sites were Y505H, N501Y, G496S, G446S, and Q498R, respectively.

[0124] An expression vector for the single-point mutant protein RBD of the SARS-CoV-2 spike protein was constructed (the method is the same as step S3, except that it is mainly achieved through a one-step single-point mutation method; the primers used are shown in Table 3), and the corresponding SARS-CoV-2 spike protein RBD single-point mutant protein was expressed and purified (the method is the same as step S4).

[0125] Table 3

[0126] G446S-F CGACTCTAAGGTGagcGGCAACTACAAC G446S-R GTTGTAGTTGCCgctCACCTTAGAGTCG G446T-F CTCGACTCTAAGGTGaccGGCAACTACAACTACC G446T-R GGTAGTTGTAGTTGCCggtCACCTTAGAGTCGAG G496S-F CACTCCAGTCTTACtccTTCCAGCCTAC G496S-R GTAGGCTGGAAggaGTAAGACTGGAGTG Q498R-F GTCTTACGGCTTCaggCCTACAAACGGC Q498R-R GCCGTTTGTAGGcctGAAGCCGTAAGAC N501Y-F CTTCCAGCCTACAtacGGCGTGGGCTAC N501Y-R GTAGCCCACGCCgtaTGTAGGCTGGAAG Y505H-F CAAACGGCGTGGGCcacCAGCCATACC Y505H-R GGTATGGCTGgtgGCCCACGCCGTTTG

[0127] The expression and purification results of the PT SARS-CoV-2 spike protein RBD point mutant protein are as follows: Figure 5 As shown.

[0128] S7. The interaction between the RBD mutant protein and hACE2 after a single point mutation of an important amino acid residue in the RBD CR3 subdomain of the SARS-CoV-2 spike protein was detected by surface plasmon resonance (SPR) technique. The effect of the single point mutation on the interaction affinity between the RBD and hACE2 of the original SARS-CoV-2 spike protein was compared.

[0129] The specific steps are the same as step S5.

[0130] Compared to the prototype SARS-CoV-2 spike protein RBD, most of the single-point mutations selected in the CR3 subdomain originated from the interaction boundary between the spike protein and hACE2 in the SARS-CoV-2 Omeprone mutant strain B.1.1.529. Replacing the corresponding subdomain of the prototype SARS-CoV-2 spike protein RBD with the CR3 subdomain of the B.1.1.529 mutant spike protein RBD increased the affinity of the chimeric mutant by approximately 20-fold (K). D =1.85±0.12nM, prototype strain K D =44±2.06nM); in contrast, most of the selected single-point mutations reduced the affinity of the corresponding RBD for hACE2, with reductions ranging from approximately 2 to 8 times: K D,Y505H =388.33±13.65nM, K D,G446S =117.33±5.86nM, K D,Q498 R = 58.6 ± 0.4 nM, K D , G496S =256.33±13 nM. The effects of some point mutations on affinity were not accurately measured in previous studies, such as the effect of the Q498R mutation. Only the N501Y mutation significantly enhanced affinity (K). D =7.39±0.14nM), which is consistent with a large number of previous studies. This reflects the accuracy and reliability of the measurement system in this experiment. Furthermore, this mutation is consistent with the effect of the entire CR3 subdomain substitution on RBD affinity, indicating the importance of the N501Y site in contributing affinity to the entire CR3 subdomain. Figure 6 As shown.

[0131] In summary, the molecular mechanism by which the CR3 subdomain contributes to affinity involves a network of polar and nonpolar interactions between key amino acid residues. A single mutation in one of these amino acid residues can disrupt the local interaction network.

[0132] S8. Based on the published analysis of the novel coronavirus, the molecular mechanism by which the CR3 chimeric mutant contributes significantly to the affinity of RBD and hACE2.

[0133] After the CR3 subdomain of the SARS coronavirus spike protein was replaced by the CR3 subdomain of the prototype SARS-CoV spike protein, the affinity of the chimeric mutant was increased by approximately 100-fold. This high affinity contrasts sharply with the relatively weak affinity of the SARS coronavirus spike protein RBD. Because the SARS coronavirus spike protein and other β-coronavirus spike protein RBD protein backbones are highly conserved, this high local affinity originates from a local interaction network formed by specific amino acid residues within the CR3 subdomain. Based on the crystal structure of the complex formed by the SARS spike protein RBD and hACE2 (pdb#2AJF, as shown in the image),... Figure 7 As shown in Figure A, in its CR3 subdomain, hACE2's Y41 and E37 form hydrogen bond networks with SARS-Cov-1's RBDT486 and Y491, respectively. On this side, we can also see that hACE2's N330 and E329 form hydrogen bonds with SARS-Cov-1's T486 and R426. On the other side, hACE2's D38 and Y41 form hydrogen bonds and hydrophobic T-shaped π-π interactions with SARS-Cov-1's RBD, respectively. In contrast, the crystal structure of the complex formed by the spike protein RBD and hACE2 of the prototype SARS-CoV-2 strain (pdb#6M0J) shows a local interaction network between two amino acid residues on both sides of the 2AJF region. The interaction network on one side consists of hACE2's Y41 and E37 residues and RBD's Y505, N501, and T500 residues, while the other side of the SARS-CoV-2 complex forms a denser interaction network, including hACE2's Q42 and D38 residues and RBD's Y449, G446, and Q498 residues. There are also hydrogen bonds formed between the two networks: G496 / RBD and K353 / hACE2. However, the replacement of the CR3 layer in the SARS-CoV-1 spike protein RBD significantly enhances the affinity of the chimeric mutant. The local interaction networks of the CR3 subdomains of the two complexes indicate that the π-π hydrophobic interaction between hACE2's Y41 residues and RBD's Y498 residues is the primary difference in interaction.

[0134] Such π-π hydrophobic interactions are also present in the RBD / hACE2 complex crystal structures (pdb#7TN0 and pdb#7XWA) of the SARS-CoV-2 Omega-3 mutant strains (B.1.1.529 and BA.5.2); and the distance between the amino acid residues Y41 / hACE2 and Y501 / RBD amino acid side chain benzene ring centers between the two molecules with π-π hydrophobic interactions is also shortened. In contrast, in the CR3 domain of coronavirus RaTG13, the density of residue interactions such as local hydrogen bonds shown above is significantly reduced. It is worth noting that Y446, which has been conserved in the RBD of the selected coronavirus spike protein, corresponds to residue F449 in the RaTG RBD. The loss of the hydroxyl group on the benzene ring also means that the hydrogen bond formed between Y449 in the RBD of other β-coronaviruses and D38 / Q42 of hACE2 no longer exists. These two important structural elements can explain why the chimeric mutant formed after the CR3 subdomain of the RaTG13 spike protein RBD replaced the prototype SARS-CoV-2 spike protein RBD almost lost its ability to bind to hACE2; for example Figure 7 As shown in Figure E.

[0135] In summary, it can be seen that in the CR3 domain: ① Especially the selected single-site mutants Y505H, N501Y, G496S, 446S, and Q498R, except for N501Y which can form a stable π-π interaction with Y41 of hACE2, the other single-site mutations will disrupt the local hydrogen bond or electrostatic interaction network between the residues on both sides of the CR3 subdomain to varying degrees, leading to a decrease in the interaction affinity between these RBD single-site mutants and hACE2. ② The π-π interaction between amino acid residues with benzene ring side chains between hACE2 and RBD is an important structural factor contributing to the high affinity of the CR3 subdomain.

[0136] To further demonstrate the importance of π-π hydrophobic interactions in the high affinity between RBD and hACE2, the crystal structure of the complex of SARS-CoV-1 spike protein RBD and the specific neutralizing antibody 80R was selected as a reference. The structure of the portion of 80R at the RBD interface is very similar to the hACE2 interface of the hACE2 / RBD complex. In the CR3 subdomain region, the hydrogen bond network is almost nonexistent; the main interaction between residues is mediated by the benzene ring stacking interaction between the Y102 residue of 80R and the Y484 residue of the SARS-CoV-1 spike protein RBD (equivalent to the Y498 residue of the SARS-CoV-2 spike protein RBD). (The distance between the pseudocenters of the benzene rings is...) The benzene ring distance between corresponding residues in SARS-Cov-1RBD / hACE2 is compared to... );like Figure 7 As shown in Figure F.

[0137] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for analyzing the affinity of the spike protein receptor-binding domain for the ACE2 receptor molecule, characterized in that, Includes the following steps: S1. By comparing the complex structure formed by spike protein RBD and ACE2 of the prototype SARS-CoV-2 and other closely related viruses, the interaction interface between spike protein RBD and ACE2 is divided into different subdomains with the convex ends and the middle concave region. Other closely related viruses refer to coronaviruses that belong to the same subgenus sarbecovirus as the prototype strain of SARS-CoV-2. S2. Using the spike protein of the novel coronavirus prototype strain as a template, at least one of the subdomains is replaced with the corresponding subdomain of the spike protein RBD of other closely related viruses to form a domain replacement chimeric protein. S3. Using the spike protein RBD protein DNA molecule of the novel coronavirus prototype strain as a backbone, an expression plasmid for replacing the chimeric protein with the aforementioned domain was constructed using molecular biology methods. S4. Expression and purification yield the domain-replaced chimeric protein, which is the chimeric mutant protein of the SARS-CoV-2 spike protein. S5. The binding kinetic parameters of the chimeric mutant protein of the SARS-CoV-2 spike protein are measured by SPR technology, and the binding kinetic parameters of the spike protein of the SARS-CoV-2 prototype are referenced to analyze the influence of each subdomain on the interaction between the spike protein RBD and ACE2. S6. Based on the analysis in step S5, the effects of different subdomains of the novel coronavirus spike protein on the RBD / ACE2 interaction were confirmed, and the key subdomain of the novel coronavirus spike protein RBD acting on the ACE2 receptor molecule with high affinity was identified. Single-point mutations were performed on key amino acid residues in the key subdomain of the prototype strain of the novel coronavirus spike protein to obtain the single-point mutant protein of the novel coronavirus spike protein RBD. S7. The binding kinetic parameters of the SARS-CoV-2 spike protein RBD single-point mutant protein are measured by SPR technology, and the binding kinetic parameters of the spike protein of the SARS-CoV-2 prototype strain are referenced to analyze the influence of subdomains and single-point mutations on intermolecular interactions. S8. Based on the analysis results of steps S1 to S7, and according to the crystallographic structure of the spike protein RBD and ACE2 complex of the prototype SARS-CoV-2 and other closely related viruses, the contribution of the high-affinity interaction subdomain to the high-affinity interaction between molecules is accurately determined, and the molecular interaction mechanism and intrinsic laws are clarified.

2. The affinity analysis method according to claim 1, characterized in that, In step S1, dividing the interaction interface between the spike protein RBD and ACE2 into different subdomains based on the two protruding ends and the middle concave region includes: identifying the subdomain that contacts the α1 helix of the receptor molecule ACE2 with the amino terminus of RBD based on the differences in key points and spatial distribution of the interaction interface between the spike protein and ACE2, according to the molecular structure of the novel coronavirus spike protein RBD / ACE2 complex protein.

3. The affinity analysis method according to claim 1, characterized in that, In step S2, replacing the subdomain with the corresponding subdomain of other closely related viral spike protein RBDs specifically involves: comparing the homologous sequences and structures of the prototype SARS-CoV-2 spike protein RBD sequences with those of other closely related viruses; selecting amino acid residues within a 4 Å distance of each RBD / ACE2 complex contact surface as important interaction discrimination sites; and, based on the differences in the corresponding amino acid residues of the corresponding subdomains of the prototype SARS-CoV-2 spike protein RBDs with those of other closely related viruses, replacing the amino acids at the important interaction discrimination sites in the subdomains of the prototype SARS-CoV-2 spike protein RBD with the corresponding amino acids of other closely related viral spike protein RBDs.

4. The affinity analysis method according to claim 1, characterized in that, In step S3, constructing the expression plasmid for the domain-replacing chimeric protein using molecular biology methods specifically includes replacing the amino acid-encoded nucleotides of the important discrimination sites in the RBD subdomain of the prototype SARS-CoV-2 spike protein with the corresponding amino acid-encoded nucleotides of the RBD of other closely related viral spike proteins using molecular biology methods.

5. The affinity analysis method according to claim 1, characterized in that, In step S4, the expression and purification of the domain-replaced chimeric protein includes: culturing cells containing the corresponding expression vector.

6. The affinity analysis method according to claim 1, characterized in that, In step S5, the analysis of the effect of each subdomain on the interaction between the spike protein RBD and ACE2 includes: comparing the affinity of the prototype SARS-CoV-2 spike protein and the SARS-CoV-2 spike protein chimeric mutant protein, and identifying the contribution of the subdomain to the affinity of RBD and ACE2.

7. The affinity analysis method according to claim 1, characterized in that, The binding dynamic parameters include the dissociation equilibrium dynamic parameter K. D .

8. The affinity analysis method according to claim 1, characterized in that, The N-terminus and / or C-terminus of the chimeric mutant SARS-CoV-2 spike protein are tagged; and / or The N-terminus and / or C-terminus of the SARS-CoV-2 spike protein RBD single-point mutant protein are tagged.

9. The application of the affinity analysis method according to any one of claims 1 to 8 in the design, screening, validation or antibody development of chemical molecular inhibitors of SARS-CoV-2.

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