Allosteric Control Overcomes Steric Limitations for Neutralizing Antibodies Targeting Conserved Binding Epitopes of the SARS-CoV-2 Spike Protein: Exploring the Intersection of Binding, Allostery, and Immune Escape with a Multimodal Computational Approach

Abstract

Understanding the atomistic basis of multi-layer mechanisms employed by broadly reactive neutralizing antibodies of the SARS-CoV-2 spike protein without directly blocking receptor engagement remains an important challenge in coronavirus immunology. Class 4 antibodies represent an intriguing case: they target a deeply conserved, cryptic epitope on the receptor-binding domain yet exhibit variable neutralization potency across subgroups F1 (CR3022, EY6A, COVA1-16), F2 (DH1047), and F3 (S2X259). The molecular basis for this variability is not fully understood. Here, we employed a multi-modal computational approach integrating atomistic and coarse-grained molecular dynamics simulations, binding free energy calculations, mutational scanning, and dynamic network analysis to elucidate how these antibodies engage the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and influence its function. Our results reveal that neutralization efficacy arises from the interplay of direct interfacial interactions and allosteric effects. Group F1 antibodies (CR3022, EY6A, COVA1-16) primarily operate via classic allostery, modulating flexibility in RBD loop regions to indirectly interfere with the ACE2 receptor binding through long-range effects. Group F2 antibody DH1047 represents an intermediate mechanism, combining partial steric hindrance-through engagement of ACE2-critical residues T376, R408, V503, and Y508-with significant allosteric influence, facilitated by localized communication pathways linking the epitope to the receptor interface. Group F3 antibody S2X259 achieves potent neutralization through a synergistic mechanism involving direct competition with ACE2 and localized allosteric stabilization, albeit with potentially increased escape vulnerability. Dynamic network analysis identified a conserved "allosteric ring" within the RBD core that serves as a structural scaffold for long-range signal propagation, with antibody-specific extensions modulating communication to the ACE2 interface. These findings support a model where Class 4 neutralization strategies evolve through the refinement of peripheral allosteric connections rather than epitope redesign. This study establishes a robust computational framework for understanding the atomistic basis of neutralization activity and immune escape for Class 4 antibodies, highlighting how the interplay of binding energetics, conformational dynamics, and allosteric modulation governs their effectiveness against SARS-CoV-2.

Keywords: Omicron variants; SARS-CoV-2 spike protein; antibody binding; binding energetics; evolutionary mechanisms; immune escape; molecular dynamics; mutational scanning; protein stability.

Figure 1

Structural organization of the RBD complexes and binding epitopes for Class 4 antibodies. (A) The structure of Class 4, group F1 CR3022 with RBD (pdb id 6YM0). The RBD is represented with wheat-colored surface, the heavy chain with orange ribbons and the light chain with red ribbons. (B) The RBD and binding epitope footprint for CR3022. The binding epitope residues are shown with blue surface. (C) The structure of Class 4 group F1 antibody EY6A bound with RBD (pdb id 7ZF3). (D) The RBD and binding epitope footprint for EY6A. (E) The structure of Class 4 group F1 antibody COVA1-16 bound with RBD (pdb id 7JMW). (F) The RBD and binding epitope footprint for COVA1-16. (G) The structure of Class 4 group F2 antibody DH1047 bound with RBD (pdb id 8DTK). (H) The RBD and binding epitope footprint for DH1047. (I) The structure of Class 4 group F3 antibody S2X259 bound with RBD (pdb id 7RAL). (J) The RBD and binding epitope footprint for S2X259.

Figure 2

RMSF profiles of RBD residues upon binding with Class 4 antibodies. (A) RMSF profiles for residues of the RBD in complexes with group F1 Class 4 antibodies. The figure compares the dynamic behavior of the RBD upon binding to CR3022 (PDB ID: 6YM0, orange), EY6A (PDB ID: 7ZF3, red), and COVA1-16 (PDB ID: 7JMW, blue). Residues within the central β-sheet structure (e.g., residues 354–358, 376–379, 394–403, 432–437, 452–454, 492–494, and 507–516) exhibit low RMSF values across all three antibodies, indicating minimal flexibility. Residues ~470–490 show elevated RMSF values, particularly for group F1 antibodies, reflecting enhanced local flexibility in this region. (B) RMSF profiles for residues of the RBD in complexes with group F2 (DH1047) and group F3 (S2X259) Class 4 antibodies. Group F2 (DH1047, PDB ID: 8DTK, orange): residues 450–470 exhibit moderately increased mobility, while the 470–490 loop shows reduced flexibility compared to group F1 antibodies. Group F3 (S2X259, PDB ID: 7RAL, blue): S2X259 also displays reduced flexibility in the 470–490 loop, consistent with its more direct engagement of the ACE2 interface.

Figure 3

Ensemble-based dynamic mutational profiling of the RBD intermolecular interfaces in the RBD complexes. Mutational heatmaps for RBD complex with group F1 CR3022 antibody (A,B), the RBD complex with group F1 E6YA antibody (C,D), and the RBD complex with group F1 COVA1-16 antibody (E,F). The mutational scanning heatmaps are shown for the interfacial RBD residues and interfacial heavy chain residues of respective Class 4 group F1 antibodies. The heatmaps show the computed binding free energy changes (in kcal/mol) for 20 single mutations of the interfacial positions. The standard errors of the mean for binding free energy changes using randomly selected 1000 conformational samples (0.06–0.12 kcal/mol) obtained from the atomistic trajectories.

Figure 4

Mutational heatmaps and epitope mapping of groups F2 and F3 of Class 4 antibodies binding to the RBD. Mutational heatmaps of the RBD binding interface residues for RBD complex with group F2 DH1047 antibody (A) and mutational heatmap of the heavy chain of DH1047 (B). The three-dimensional structures of the DH1047 complex with RBD (C), and a detailed view of the RBD, the epitope, and binding hotspots for DH1047 (D) (PDB ID: 8DTK). Mutational heatmaps of the RBD binding interface residues for RBD complex with group F3 S2X259 antibody (E) and mutational heatmap of the heavy chain of S2X259 (F). The three-dimensional structures of the S2X259 complex with RBD (G), and a detailed view of the RBD, the epitope, and binding hotspots for S2X259 (H) (PDB ID: 7RAL). The RBD is represented with wheat-colored surface. The epitope sites are highlighted with blue surface and binding hotspots with orange surface.

Figure 5

The ensemble-averaged SPC centrality (A) and the average Z-score of ASPL over mutational scan (B) for the RBD residues for Class 4 group F1 antibody complexes. CR3022 with RBD, pdb id 6YM0 (orange filled bars), EY6A with RBD, pdb id 7ZF3 (magenta filled bars), and COVA1-16 with RBD, pdb id 7JMW (green filled bars). (C) Structural mapping of allosteric network centers for Class 4 group F1 CR3022 antibody with RBD. (D) Structural mapping of allosteric network sites for Class 4 group F1 COVA1-16 antibody with RBD. The heavy chain is represented with orange ribbons, the light chain with red ribbons. The binding epitope residues are shown with blue surface, while binding hotspots are in orange and the allosteric residue interaction network with high SPC and Z-score ASPL values is represented with wheat-colored spheres.

Figure 6

The ensemble-average SPC centrality (A) and the average Z-score of ASPL over mutational scan (B) for the RBD residues for Class 4 group F2 antibody complexes. DH1047 with RBD, pdb id 8DTK (orange filled bars) and group F3 antibody complex S2X259 with RBD, pdb id 7RAL (magenta filled bars). (C) Structural mapping of allosteric network sites for Class 4 group F2 antibody complex DH1047 with RBD, pdb id 8DTK. (D) Structural mapping of allosteric network sites for the Class 4 group F3 antibody complex S2X259 with RBD, pdb id 7RAL. The heavy chain is shown with orange ribbons, the light chain with red ribbons. The binding epitope residues are shown with blue surface, while binding hotspots are in orange and the allosteric residue interaction network with high SPC and Z-score ASPL values is represented with wheat-colored spheres.