Structure of de Novo Designed Coiled-Coil Protein Origami Triangle
Coiled-coil protein origami (CCPO) uses modular coiled-coil (CC) building blocks and topological principles to design polyhedral structures distinct from natural globular proteins. The desired shape is defined through the topological arrangement of parallel and/or anti-parallel CC dimers arranged into a precisely defined sequential order, based on the underlying mathematical rules. CC building blocks associate according to well-defined pairing rules with combination of hydrophobic and electrostatic interactions at the heptad positions a/d and e/g, respectively of the heptad repeat. While the CCPO strategy has proven successful in designing diverse protein topologies, no high-resolution structural information has been available for these novel protein folds, due to the high flexibility and small size of these structures.
Here we report the first high-resolution crystal structure of a single-chain CCPO in the shape of a triangle. While neither cyclization nor the addition of nanobodies enabled crystallization, it was ultimately facilitated by using shorter linkers between peptides and the inclusion of natural CC homodimer - GCN2. The crystal lattice exhibited a considerable number of crystal contacts, and we hypothesize that the dense packing of triangular molecules is responsible for the high-resolution diffraction observed. The structure is consistent with the proposed triangular design, including an internal cavity. Individual CC dimers are well resolved in the structure and show the expected packing interactions between hydrophobic residues at positions a/d. The triangle is additionally stabilized by side-chain interactions between neighboring segments at each vertex, and the crystal structure also aligns with the protein in solution and fits well into the ab initio protein envelope derived from SAXS data.
A closer inspection of the structure revealed that the chain forms a relatively shallow protein knot, known as the trefoil-type knot. To our knowledge, this is the smallest knot in a designed protein. Interestingly, AlphaFold2 is unable to predict the fold of the triangle (and other CCPO structures), most likely due to the complex folding topology and absence of this type of fold in structural databases.
In conclusion, the structural validation of the modular CC-based protein design provides essential molecular insights into CCPO strategy. These findings advance our understanding of the molecular mechanisms underlying CCPO folding and stability and contribute to developing new CC-based and knotted synthetic assemblies. The authors of this research are members of the Department of Synthetic Biology and Immunology at the National Institute of Chemistry Tadej Satler, San Hadži (employed at FKKT, UL) and Roman Jerala.
Link to the article: https://pubs.acs.org/doi/10.1021/jacs.3c05531