The tertiary structure of the SARS-CoV spike protein bound to ACE2 (Song et al. 2018, Wan et al. 2020), obtained from RCSB (PDB ID: 6ACG), and the tertiary structure of the SARS-CoV RBD bound to 80R (PDB ID: 2GHW) were superimposed, and the primary sequences were aligned (Fig. 2). A structural model for the SARS-CoV-2 RBD was constructed using SWISS-MODEL and superimposed by sequence alignment and tertiary structures (Fig. 2). In the secondary structures of the spike proteins, helices are shown in red, strands are shown in yellow and loops are shown in green; ACE2 is brown and 80R is cyan. I found that the 80R antibody and ACE2 bind in the same location, which perfectly explains how the antibody prevents infection by competing with ACE2. The structure of the SARS-CoV-2 spike protein (PDB ID: 6VSB) RBD has many loops missing (data not shown). Because these are essential for binding to ACE2, the model was constructed with a valid structure to run docking experiments. Furthermore, glycans in the PDB 6VSB show no interference with 80R binding even in predicted sites but not in the crystal structure (Fig. 2).

Sequence alignments between SARS-CoV RBD and SARS-CoV-2 RBD were built in UCSF Chimera. There are many sequence differences between SARS-CoV and SARS-CoV-2 in the 80R-RBD interface, which explains why the 80R antibody binds with high affinity to the spike protein of SARS-CoV but not to the spike protein of SARS-CoV-2. In the SARS-CoV-2 RBD, polar residues are replaced by neutral residues, which disrupts the binding interactions between 80R and the RBD in the SARS-CoV-2 spike protein. Insertion of a glycine residue at position 482 also twists one of the interacting loops located at E484, which then clashes with the 80R antibody in the superimpositions. To avoid this clash and allow better antibody binding to the RBD in SARS-CoV-2, the 80R partner residue should be replaced by a different residue. I carefully selected six alternative residues, introduced these one at a time, and ran docking experiments using Rosetta Dock (Lyskov and Gray 2008, Lyskov et al. 2013) to explore the new interactions (Fig. 3). Aromatic-aromatic interactions between residues that are within 8 Å of each other are very important for protein structure and protein-protein recognition (Lanzarotti et al. 2011). According to the structural analysis in Chimera, there are many aromatic residues in the RBD of SARS-CoV-2 and I therefore introduced aromatic residues into 80R to match the RBD aromatic residues. My strategy was to thus increase aromatic-aromatic interactions, avoid the clash with E484 and maintain the solubility of the antibody by introducing substitutions, without introducing major changes in the 80R antibody. In support of my findings for the 80R antibody epitope, an epitope for another SARS-CoV-2 antibody (CR3022) was recently reported to contain many aromatic residues (Yuan et al. 2020).

I made a structural model of mutated 80R with SWISS-MODEL and positioned this model close to the RBD region. I used Rosetta Dock in Stampede2 supercomputer to run docking experiments with the following mutations in 80R R102F, S103F, R152F, S186F, T206G, S210F and N227F. These mutations were intended mainly to increase aromatic-aromatic interactions and to avoid contact with E484 (T206G) (Fig. 3). The following aromatic interactions were achieved: F102-F103-Y505, F186-F456, F152-F210-F486, and F227-Y449. These interactions increased the binding affinity between the two partners, as demonstrated by the funnel type charts from Rosetta Dock results (Fig. 4), which show how the structures converge to the lowest possible energy state compare to wild type. Furthermore, PDBEPISA (Krissinel and Henrick 2007) server was used to get the energies of the complexes with satisfactory results for the new mutated 80Rm. Finally, the Therapeutic Antibody Profiler (TAP) (Dunbar et al. 2016) tool was used to analyze the developability of this new mutated antibody and it shows all green flags for five important characteristics to take in consideration such as hydrophobicity and charges.

Similarly, m396 antibody that neutralizes SARS-CoV binding to its RBD domain was structurally analyzed and five mutations were introduced. In this case, the analysis pointed to electrostatic interactions to maximize and one aromatic. Therefore, five mutations were introduced: in the heavy chain T52F, I56E, N58K, Q61E; and in the light chain S94E. A structure with these mutations was put close to SARS-CoV-2 RBD and docking experiments were run in Stampede2. The results (Fig. 5) show that these mutations improve binding to the level of binding seen with SARS-CoV and wild type antibody (Fig. 6). m396 mutant shows formation of 9 new salt bridges which provide great binding to the target SARS-CoV-2 RBD (data not shown). Energy analysis from PDBEPISA server shows that binding is improved significantly compared to a wild type m396 and SARS-CoV-2 RBD. Furthermore, the developability flags show green even though one is yellow but just for a short margin which may be addressed after testing in wet lab experiments if it is an issue.