De novo protein design field explores novel protein folds that have not undergone evolutionary sampling. Despite the significant progress, extensive experimental validation is still required to identify the sequences with desired structure and function. An alternative strategy to protein scaffold design is based on modular building blocks with a well-understood sequence-structure relationship. Coiled-coil protein origami design strategy, coiled-coils are used as modular building blocks to design protein nanostructures. The desired shape is defined through the topological arrangement of parallel and/or anti-parallel coiled-coil dimers arranged into a precisely defined sequential order, based on the underlying mathematical rules. Although the coiled-coil protein origami has proven as a robust strategy for the design of various protein topologies, no high-resolution structural information is available for these structures. The main difficulty concerns the high flexibility and small size of origami structures, which makes them challenging to study using high-resolution methods, such as cryo-electron microscopy and x-ray crystallography.
Here we tackled the challenge of structural characterisation of protein origami structures employing various methods. Our efforts resulted in a successful determination of a high-resolution structure of a coiled-coil protein origami triangle. We prepared stabilized protein origami triangles by using enhanced variants of the peptide segments. We also reduced the flexibility of the protein fold by using shorter linkers connecting these peptide segments. Our study showed that protein origamies additionally stabilized through covalent linking of the N- and C-termini, which we achieved by using split-inteins or an enzyme tyrosinase. Furthermore, we successfully prepared various protein complexes from protein origami and either nanobodies or legobody scaffold. These complexes were designed to facilitate protein crystallization or enhance particle size and contrast for structural analysis using a cryo-electron microscope. For complex preparation, we used both known coiled-coil binding nanobodies and nanobodies for which we transplanted an epitope onto the coiled-coil motif. We established a modular system for epitope transplantation of the Nb49 nanobody and the Ib3 intrabody, and confirmed their efficiency through several different protein constructs. By employing more stable peptide segments, shorter linkers, and integrating the natural homodimeric GCN2 peptide pair, we engineered a coiled-coil triangle named TRI-4SHbGCN, that successfully crystallized and diffracted in high resolution. Crystal structure confirmed designed triangular shape with the large central cavity and is additionally stabilized by side-chain interactions between neighbouring segments at each vertex. The orthogonal peptide segments associate with their pair and remain unperturbed in the context of protein origami structure. Interestingly, the polypeptide chain folds into a trefoil-type protein knot topology. The structure validates the modular CC-based protein design strategy, providing molecular insight underlying CCPO stabilization and new opportunities for improving the design.
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