Figure 6. Affinity driving mutations in Omicron RBD have previously been identified by in vitro evolution for tighter binding.
(A) Analysis of the occurrence and prevalence of Omicron variant mutations. The background is coloured according to S-protein functional domains: NTD domain (AA 12 – 306; orange), RBD domain (AA 318 – 514; green), Furin cleavage site and its proximity (AA 655 – 701; blue). The four positions critical for the high affinity of RBD-62 are highlighted in bold. Mutation frequencies within individual lineages are denoted in green (100–75 %), blue (75–50 %) and magenta (50–25 %). Information about the distribution and frequency of S-protein mutations and the spatiotemporal characterization of SARS-CoV-2 lineages was retrieved from www.outbreak.info (Mullen et al., 2020) and Gisaid database (Elbe and Buckland-Merrett, 2017).* Same evolutionary origin, a Number of evolutionary non-related lineages with given or similar mutation (Zahradnik et al., 2021c), b log(10) number of the observed Omicron mutation at the given position as determined on 14.11.2021, c same as b but total log(10) number of changes at the given position. d fold-change in binding as determined by yeast-surface display. (B) Comparison of fold change in binding affinity among selected mutations and their combinations as determined by titrating ACE2 on yeast surface displayed RBD mutations. Values are fold-change relative to the original strain. For Omicron, yeast titration is denoted in violet, SPR (this study) is black, SPR as determined in (Cameroni et al., 2021) is grey and ELISA as determined in (Schubert et al., 2021) is in orange. Data denoted by black dots have been reported previously (Zahradnik et al., 2021b). (C) RBD-62 (blue)/ACE2 (green) structure (PDB: 7BH9) overlaid on Omicron RBD structure (orange) as determined bound to Beta 55. All Omicron mutations are shown, overlaid on relevant RBD-62 mutations. (D) Electrostatic potential surface depictions calculated using the APBS plugin in PyMol for left to right: early pandemic Victoria RBD showing the ACE2 interacting surface, ACE2 showing surface that binds the RBD, Beta RBD ACE2 interacting surface, RBD-62 ACE2 interacting surface, Omicron RBD ACE2 interacting surface. Blue is positive and red negative potential (scale bar shown above).