Designed protein self-assembly by autocatalytic isopeptide bond formation

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Owing to their structural diversity, proteins perform a variety of functions in living systems from catalysis to serving as nature’s building block for the assembly of dynamic materials and molecular machines. Inspired by these natural examples, researchers have sought ways to design protein assemblies from discrete oligomeric structures to higher-order structures (e.g., 1D fibers, 2D arrays, and 3D lattices) following different non-covalent and covalent assembly strategies. Isopeptide bonds (IPB) are a particularly intriguing example of how nature utilizes covalent bonds to build higher-order protein structures and equip them with exceptional stability. Yet, these natural IPB-forming systems are limited to few protein classes. Previously, engineered variants of these natural IPB-forming systems have been used as genetic fusion tags to drive autocatalytic protein assembly. But these systems are derived from pathogenic bacteria and always leave a molecular scar, which can prompt unwanted immune responses in potential therapeutic applications. Being able to rationally design autocatalytic IPB into a protein-protein interface of choice would thus be highly desirable. This project aims to i) rationalize de novo IPB design in proteins that are structurally unrelated to natural IPB-forming systems and ii) utilize the de novo design of IPB for the assembly of discrete and higher-order protein assemblies. To this end, the homodimeric four-helix bundle protein Rop will be used as proof-of-principle. The four-helix architecture represents a common motif that is often observed independently and as a subunit of larger protein folds and is structurally unrelated to any natural IPB-forming system. A combined approach of computational design and directed evolution will be used to establish homo- and heterodimeric protein assemblies with different numbers of IPB, thereby providing tunable levels of assembly stabilization. Using these discrete assemblies as building blocks, IPB formation will be used to build higher-order structures with high levels of covalent interconnectivity as well as 1D assemblies (i.e., linear protein polymers), both of which are expected to possess exceptional structural stability. Finally, the IPB design principles established in the initial phase of the project will be transferred to other proteins to test the generality of the approach. This could lead to interesting new applications. For instance, by combining the structure-stabilizing features of IPB with orthogonal and switchable protein assembly strategies (e.g., metal-templated assembly or disulfide bonds), stable but stimuli responsive nanostructures with potential therapeutic relevance are envisioned. Overall, the proposed project will demonstrate the de novo design of IPB as a novel means to establish structure-stabilizing covalent interactions that will drive the next generation of protein assemblies.