Interfacial subregions of SARS-CoV-2 spike RBD to hACE2 affect intermolecular affinity by their distinct roles played in association and dissociation kinetics

Figures

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  Fig. 1. Scheme of the spatially distributed subdomains of the SARS-CoV-2 spike RBD and designment of the RBD chimeric mutants of specific swapped subdomains to disclose structural determinants governing SARS-CoV-2 RBD/hACE2 interaction. A WT SARS-CoV-2’s spike RBD, especially RBM can be spatially partitioned into three subdomains owing to their specific hACE2 contact resides and spatial arrangement. Similar RBD structural organizational patterns are detected in SARS-CoV-2 Omicron (B.1.1.529), SARS-CoV-1 and bat coronavirus RaTG13; The RBD structures as presented above are from spike RBD/hACE2 crystal structures with PDB# 6M0J, 7TN0, 2AJF, and 7DRV31,37,44,51. B SPR measurement of RBDs’ binding to hACE2(1-740)-FC immobilized on Series S Sensor Chip (Protein A), which cover those from above sarbecovirus species, but not that of RaTG13. The equilibrium dissociation constants (KD) from triplicate measurements were calculated with BIAcore@ 8 K Evaluation Software (GE Healthcare) by fitting to a 1:1 Langmuir binding model. C RBD protein sequence alignment between WT SARS-CoV-2, Omicron (B.1.1.529), SARS-CoV-1 and bat coronavirus RaTG13. The secondary structures as discovered in WT SARS-CoV-2…

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  Fig. 2. Affinity measurement of the impact on the SARS-CoV-2 RBD/hACE2 interaction by swapped RBD subdomains from RaTG13, SARS-CoV-1 and Omicron (B.1.1.529). A WT SARS-CoV-2’s CR1 subdomain was swapped and the resulting chimeric mutants’ binding to hACE2(1-740) was measured by SPR. Chimeric swapped CR1 mutant from SARS-CoV-1 could not be expressed, which was replaced by a SARS-CoV-2 RBD chimera containing SARS-CoV-1 RBM replacement, binding affinity measurement of which reflects inherent coordination among CR1, CR2 and CR3 subdomains. B WT SARS-CoV-2’s CR2 subdomain was swapped and the resulting chimeric mutants’ binding to hACE2(1-740) was measured by SPR. C WT SARS-CoV-2’s CR3 subdomain was swapped and the resulting chimeric mutants’ binding to hACE2(1-740) was measured by SPR.

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  Fig. 3. Scheme of the natural mutations distributed on RBD, especially on the RBM of SARS-CoV-2 and RBD/hACE2 binding affinities’ comparison among representative world-wide circulating SARS-CoV-2 VOCs. A Top view of the respective SARS-CoV-2 VOCs’ RBM with key residues in contact with hACE2 of WT SARS-CoV-2(top left) or corresponding mutated residues from other VOCs colored accordingly. Also array of key mutated residue locations clustered from all selected VOCs are projected onto the same WT SARS-CoV-2 RBD structure (2nd image on top left). Protein structures of wild type, Alpha(B.1.17), Beta(B.1.351), Gamma(P.1), Delta(B.1.617.2), and Omicron (B.1.1.529, BA.5.2) RBDs are referred to PDB# 6M0J, 7MJN, 7V80, 7NXC, 7WBQ, 7TN0, and 7XWA respectively31,47,50,51,66,67. B Scheme of natural mutations distributed along the SARS-CoV-2 VOCs’ primary RBD protein sequence. For WT SARS-CoV-2 RBD, the RBM sequence coverage is labeled by a two-way arrow. For Omicron (BA.5.2), additional natural mutations to those of Omicron (B.1.1.529) are displayed in deep blue and retro-mutated residues of R493Q and S496G in RBM are colored in green. C RBD binding affinities to hACE2 are compared among selected…

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  Fig. 4. SPR scanning to differentiate selected single residues’ impact onto the SARS-CoV-2 RBD/hACE2 interaction. A Cartoon shows naturally occurring key RBD mutations in SARS-CoV-2 Omicron (B.1.1.529 and BA.5.2), and distinct residues of SARS-CoV-1 and RaTG13 from those of WT SARS-CoV-2 spike RBD, with their respective locations projected onto the WT SARS-CoV-2 RBD structure (PDB# 6M0J). B SPR measurements of binding affinity of single SARS-CoV-2 RBD mutants of RBM region interacting with hACE2. For each single mutant, the equilibrium dissociation constants (KD) from triplicate measurements were analyzed with BIAcore@ 8 K Evaluation Software (GE Healthcare) by fitting to a 1:1 Langmuir binding model. Purple, red and yellow colors represent mutants distributed in CR1, CR2 and CR3 subdomains respectively as presented in cartoon A. C SPR measurement of binding affinities of single SARS-CoV-2 RBD mutants of non-RBM region for hACE2.

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  Fig. 5. Binding kinetics analysis discloses distinct impact on RBD/hACE2 association and dissociation kinetics by CR2 and CR3 subdomains respectively. A Association kinetics profiles of RBD/hACE2 complex formation by SARS-CoV-2 chimeric mutants of swapped CR1/2/3 and single residue mutants reveal significant CR2’s impact on RBD/hACE2’s initial contact with hACE2, in which long range electrostatic force are involved. The mutations’ impact as compared to WT SARS-CoV-2 RBD was quantified as the ratio of ka(RBD mutant) to ka(wild type). For the swapped CR2 mutants from SARS-CoV-1 and Omicron (B.1.1.529 & BA.5.2), the mutated residues are marked and positively charged residues are labeled in orange. B Dissociation kinetics of RBD/hACE2 complex formation by SARS-CoV-2 chimeric mutants of swapped CR1/2/3 and single key residue mutants reveal CR3’s significant impact on RBD/ hACE2 complex stability. The mutations’ impact as compared to WT SARS-CoV-2 RBD was quantified as the ratio of kd(wild type) to kd(RBD mutant). C Relative binding affinity change due to swapped CR1/2/3 subdomains or single key residue mutations as compared to WT SARS-CoV-2 RBD interacting with hACE2. The mutations’ impact is…

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  Fig. 6. Structural reference of selected swapped CR1, CR2 and CR3 subdomains of strong affinities and a phase specific binding mode model attributing spike RBD/hACE2 affinity to distinct effectors in association and dissociation kinetics. A Structural reference of selected swapped CR1/CR2/CR3 subdomains of strong affinities based on SPR measurements in this study. For each subdomain contact interface’s structural display, key residues are marked and critical inter-residue bonding such as π-π interactions or hydrogen bonds were indicated by two-way arrows, with hydrogen-bonded atoms or pseudoatom centers of phenolic group of involved residues shown as colored spheres; The interaction networks can be spatially distinguished into separate polar interaction networks outlined by dashed ellipse. In the left top figure, the two bonding interaction networks of CR3 interface draw hACE2’s N terminal α1 helix towards RBD side by side (indicated by two parallel arrows), while in left bottom figure, two bonding networks linked by inter-group bonding generate a stronger combined pulling force to make RBD/hACE2 complex more stable. PDB# of the reference structure or modeling information for each…