Fig. 1

Workflow for computational design of trivalent anti-SARS-CoV-2 microproteins. a Structure-guided computational design of trivalent microproteins to geometrically match the three binding sites of the trimeric S protein. The trimerization scaffold, linker, and monovalent binder are shown in blue, green, and gray, respectively. RosettaRemodel was used to design linkers connecting the C-terminus of the monovalent binder and the N-terminus of the trimerization scaffold. b Molecular dynamics (MD) evaluation of the trivalent constructs. Binding free energies of the monomers were estimated to assess trimerization tendency using the MM/GBSA method. Free energy landscapes were constructed to study the possible distributions of the trimer conformations. c Experimental verification of trivalent constructs using size-exclusion chromatography and Native-PAGE. d Functional tests of the top-ranked constructs. Binding affinity was measured by BLI, and pseudovirus neutralization assays were performed to determine the neutralizing activity against SARS-CoV-2