In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission. The ability to predict mutations in SARS-CoV-2 that cause increased infectivity or immune evasion would be valuable in development of vaccines and therapeutics, and prioritizing viral variants for monitoring and containment. Using rapid in vitro evolution, we identify mutations in SARS-CoV-2 receptor binding domain (RBD) that markedly increase binding of RBD to ACE2. One mutation in particular, Q498H, causes a substantial increase in binding affinity, and we uncover the mechanism for this by structural determination. We show addition of Q498H increases binding of some SARS-CoV-2 variant RBDs but find Q498H is incompatible with the common N501Y mutation. RBD variants with N501Y can instead access similar bonding and affinity increases by acquiring the alternative Q498R mutation. The Q498R/N501Y combination mutations, and Q498H, also enable de novo high affinity binding of SARS-CoV-2 RBD to rat ACE2. Our data show SARS-CoV2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in new SARS-CoV2 variants, where they have the potential to contribute to increased variant transmissibility and potentially facilitate cross-species transmission.