Preorganized cyclic modules facilitate the self-assembly of protein nanostructures

The rational design of supramolecular assemblies aims to generate complex systems based on the simple information encoded in the chemical structure. Programmable molecules such as nucleic acids and polypeptides are particularly suitable for designing diverse assemblies and shapes not found in nature. Here, we describe a strategy for assembling modular architectures based on structurally and covalently preorganized subunits. Cyclization through spontaneous self-splicing of split intein and coiled-coil dimer-based interactions of polypeptide chains provide structural constraints, facilitating the desired assembly. We demonstrate the implementation of a strategy based on the preorganization of the subunits by designing a two-chain coiled-coil protein origami (CCPO) assembly that adopts a tetrahedral topology only when one or both subunit chains are covalently cyclized. Employing this strategy, we further design a 109 kDa trimeric CCPO assembly comprising 24 CC-forming segments. In this case, intein cyclization was crucial for the assembly of a concave octahedral scaffold, a newly designed protein fold. The study highlights the importance of preorganization of building modules to facilitate the self-assembly of higher-order supramolecular structures. Modular protein cages can self-assemble from multiple preorganized modules. We implemented an intein-mediated cyclization that enhanced the structural stability and definition of protein-protein interaction surfaces.