Recombinant Engineering of SARS-CoV-2 Spike and N proteins

National Institute Of Allergy And Infectious Diseases

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Abstract

We have successfully engineering and produced a number of SARS-CoV-2 proteins, subunits, and single domain antibodies directed against the RBD. These purified proteins have provided the materials for obtaining high resolution crystal structures of four of these sybodies in complex with the Spike receptor binding domain (RBD) and one unliganded. In addition, we have addressed structural aspects of the sybody interactions with the complete Spike protein, using cryo-electron microscopy. We have extended our binding studies to evaluate the effects of SARS-CoV-2 variants of concern on the recognition by these sybodies. We have performed in silico mutagenesis of the RBD and molecular modeling to explain the results of our binding. A paper describing the engineering, purification, binding, X-ray and cryo-EM structures has been submitted and is currently under revision. The details of the nature of the interaction footprint of the sybodies explains their ability to inhibit virus infection. Preprints describing these findings have been posted on BioRXiv. The X-ray structures have been deposited and are available at the RCSB Protein Data Bank (PDB) under accession numbers: 7KGK, 7KGJ, 7KLW, 7MFU, and 7MFV with resolutions ranging from 1.7 to 2.6 Angstrom. The cryo-EM structures are available at the Electron Microscope Data Base (EMDB) under accession numbers: EMD-24105 and EMD-24106 with overall map resolution of 3.02 and 3.34 Angstrom respectively. Detailed molecular models derived from the cryo-EM maps are available at the PDB under numbers 7N0G and 7N0H.