Fig. 1: Structure-guided design, production, and characterization of the mutI-tri-RBD.

a A schematic illustration of the mutI-tri-RBD and homo-tri-RBD design schemes. The RBD region comprising the residues 319–537 was truncated from the S protein, and three truncated RBDs were connected end-by-end to construct the trimeric forms of RBD. In mutI-tri-RBD, three RBDs were individually derived from three different circulating SARS-CoV-2 strains, i.e., the prototype, Beta and Kappa. In homo-tri-RBD, the three RBD units were all truncated from the prototype strain. In the upper subfigure, the S1 and S2 subunits of the S protein, as well as NTD, RBD, SD1, and SD2 in the S1 subunit, are marked. The lower subfigure displays the natural trimeric arrangement of RBDs in the native structure of S trimer. The arrows indicate the direct connections of the N- and C- terminals between different RBDs. b Structural modeling and MD simulation of the designed homo-tri-RBD (upper subfigure) and mutI-tri-RBD (lower subfigure). Time-evolution of the C_α root-mean square deviation (RMSD) of the modeled structure during MD simulation, as well as several snapshot conformations in the simulation, is displayed. c SDS-PAGE profiles of increasing amounts of the recombinant mutI-tri-RBD and homo-tri-RBD proteins expressed by HEK293T cells. d Molecular weight of mutI-tri-RBD determined by MALDI-TOF MS. e Molecular weight of mutI-tri-RBD after deglycosylation determined by UPLC-MS. f Secondary structure contents of mutI-tri-RBD protein analyzed by circular dichroism spectrometry. g Left: the proportions of free sulfhydryl for all the cysteine residues in mutI-tri-RBD. Right: the disulfide linkages in the recombinant mutI-tri-RBD protein detected by liquid chromatography-mass spectrometry. Only the disulfide bonds in one RBD unit are listed. h Differential scanning calorimetry thermograms of the recombinant mutI-tri-RBD protein. i The binding capability of the designed mutI-tri-RBD and homo-tri-RBD proteins with two anti-RBD monoclonal nAbs, i.e., MM43 and R117, evaluated by ELISA. As controls, the binding activities with the monoclonal nAbs for the monomeric his-tagged RBDs from the prototype, Beta, and Kappa SARS-CoV-2 strains were also measured. j The binding profiles of the recombinant mutI-tri-RBD with hACE2 detected by surface plasmon resonance assay.