Crystal Structure of de Novo Designed Coiled-Coil Protein Origami Triangle

Abstract

Coiled-coil protein origami (CCPO) uses modular coiled-coil building blocks and topological principles to design polyhedral structures distinct from those of natural globular proteins. While the CCPO strategy has proven successful in designing diverse protein topologies, no high-resolution structural information has been available about these novel protein folds. Here we report the 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 the inclusion of a GCN2 homodimer. Triangle edges are formed by the orthogonal parallel coiled-coil dimers P1:P2, P3:P4, and GCN2 connected by short linkers. A triangle has a large central cavity and is additionally stabilized by side-chain interactions between neighboring segments at each vertex. The crystal lattice is densely packed and stabilized by a large number of contacts between triangles. Interestingly, the polypeptide chain folds into a trefoil-type protein knot topology, and AlphaFold2 fails to predict the correct fold. The structure validates the modular CC-based protein design strategy, providing molecular insight underlying CCPO stabilization and new opportunities for the design.

Preorganized cyclic modules facilitate the self-assembly of protein nanostructures

“…In this regard, we have already observed that CCPO structures seem inherently difficult to crystalize or vitrify; only recently an X-ray structure of the CCPO triangle has been determined based on the fortuitous packing of CC modules into a crystal lattice. 77 We therefore resorted to using small-angle X-ray scattering (SAXS) ( Fig. 2D, I and N , and Table 1 ) a method that allows determination of the molecular shape directly in solution.…”

“…5,10,86 Interestingly, algorithms, such as AlphaFold2, have not been able to predict the structure of CCPOs. 77…”

Comparative Simulative Analysis and Design of Single-Chain Self-Assembled Protein Cages

“…Using oxDNA, a coarse-grained model was proposed, which has been applied to characterize the structures of DNA star-tile-based nanocages and the self-assembly of DNA origami and single-chain-knotted DNA nanostructures . Recent high-resolution determination of a CCPO triangle confirmed the structure according to the design . While the difficulties in the determination of the high-resolution structure of CCPOs suggested that the flexibility of linkers may introduce high flexibility, stronger evidence could be provided by a simulation.…”

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure

“…De novo protein design allows for the creation of protein structures that are distinct from those found in nature, assisted with recent development of machine learning. 1 Despite leveraging natural building blocks like coiled-coils, 2 achieving entirely novel topologies remains a significant challenge. Designing functional proteins presents another formidable task.…”