Abstract
Circular tandem repeat proteins (‘cTRPs’) are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.
Highlights
Circular tandem repeat proteins (‘circular tandem repeat proteins (cTRPs)’) are de novo designed protein scaffolds that contain repeated protein sequences and structural motifs and form closed circular structures
We have previously described the creation of an array of circular tandem repeat proteins (‘cTRPs’) that are constructed from repeated two-helix bundles and that display a wide range of sizes and symmetries[5,13]
When applying this approach to the design of closed tandem repeat proteins with increased thickness (‘thick(er) cTRP (tcTRP)’ or ‘thick(er) circular tandem repeat proteins’), we identified designs with repeats corresponding to right-handed helical bundles as displaying favorable predicted folding energetics for their designed backbone topologies, and well-formed energy funnels when subjecting their sequences to unbiased fold predictions in Rosetta
Summary
Circular tandem repeat proteins (‘cTRPs’) are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. We have previously described the creation of an array of circular tandem repeat proteins (‘cTRPs’) that are constructed from repeated two-helix bundles and that display a wide range of sizes and symmetries[5,13] The largest of these constructs was used to create symmetric protein nanoparticles that can incorporate a variety of functional protein via covalent attachment around their periphery. CTRPs that display multiple binding domains can display significant avidity effects (due to multimeric display of those domains around the molecular surface) and have been used to create novel formulations of immunological stimulatory and signaling molecules that can be used for biotechnology applications such as therapeutic T-cell manufacture While these cTRP constructs have many favorable properties, we have previously noted that their ability to self-assemble from smaller subunits is compromised by relatively limited contacts and small surface areas that are involved in packing between repeats and subunits. We hypothesized that a new generation of computationally designed cTRP proteins with increased repeat size (corresponding to longer secondary structural elements that increase both particle thickness and the number of buried contacts between repeats and protein subunits) would improve both the energetics and the stoichiometric control of self-association
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