Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning
Wenhao O. Ouyang, Timothy J.C. Tan, Ruipeng Lei, Ge Song, Collin Kieffer, Raiees Andrabi, Kenneth A. Matreyek, Nicholas C. Wu
Abstract
Increasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the amino-terminal domain (NTD) has been long overlooked because of the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD but also provides invaluable insights into S-based immunogen design.