The sequence of human ACE2 is suboptimal for binding the S spike protein of SARS coronavirus 2

Erik Procko

were also enriched at buried positions where they will change local packing (e.g. A25V, 172 L29F, W69V, F72Y and L351F). The selection of ACE2 variants for high binding signal 173 therefore not only reports on affinity, but also on presentation at the membrane of folded 174 structure recognized by SARS-CoV-2 S. The presence of enriched structural mutations in 175 the sequence landscape is especially notable considering the ACE2 library was biased 176 towards solvent-exposed positions. 177 Thirty single substitutions highly enriched in the nCoV-S-High sort were validated by 178 targeted mutagenesis (Fig. 5) . Binding of RBD-sfGFP to full length ACE2 mutants increased 179 compared to wild type, yet improvements were small and were most apparent on cells 180 expressing low ACE2 levels (Fig. 5A) . To rapidly assess mutations in a format more 181 relevant to therapeutic development, the soluble ACE2 protease domain was fused to 182 sfGFP. Expression levels of sACE2-sfGFP were qualitatively evaluated by fluorescence of 183 the transfected cultures (Fig. 6A) , and binding of sACE2-sfGFP to full length S expressed at 184 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.16.994236 doi: bioRxiv preprint the plasma membrane was measured by flow cytometry (Fig. 6B) . A single substitution 185 (T92Q) that eliminates the N90 glycan gave a small increase in binding signal (Fig. 6B ). 186