Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

There is considerable interest in the design of novel protein assemblies, for example, to develop cryogenic electron microscopy (cryo-EM) scaffolds to aid in structure determination (1, 2) and protein nanoparticle vaccines (3, 4). Many studies have utilized 1) genetic fusion of α-helices at N and C termini or 2) protein interface design to drive assembly of symmetric protein homo-oligomers into a larger symmetric nanoparticle or lattice. The genetic fusion route was originally demonstrated with the creation of a tetrahedral protein nanocage and fiber (5) and has since been used to generate two-dimensional (2D) layers (6), a porous cube (7), additional tetrahedra (8, 9), octahedra (10), and icosahedra (11, 12). Strengths of the fusion approach are that it is relatively straightforward, not inherently destabilizing, and guarantees the intended stoichiometry and specificity between fusion partners. Despite its simplicity and success, several aspects of genetic fusion complicate its use. Genetic fusion is best applied with α-helical termini, as this imparts some rigidity between the fused domains, but termini are not always α-helical or physically accessible. Furthermore, the number of possible sequence alignments between any given pair of building blocks is finite and this, in turn, limits the number of geometric solutions for a desired symmetric architecture. This can complicate or preclude the use of fusion methods in assembly design, especially when a particular protein building block is required for a downstream application. Finally, flexibility is often introduced at the point of fusion, even with α-helical linkers and certainly with disordered linkers. In the best cases, model deviations are subtle (8, 13); however, varying levels of unintended assembly products are also commonly observed. In contrast, the noncovalent protein interface design approach bypasses considerations of termini and their alignment. Interface design is compatible with the free rotation and translation of building blocks along their axes (14), so the set of valid geometric solutions is technically unlimited. The drawback to interface design is that it requires the creation of a new soluble and specific interface without destabilizing either protein. Like fusion, the interface design route to the protein nanomaterial production has achieved considerable success (4, 15–20).Among applications for genetic fusion is the creation of cryo-EM scaffolds (1, 2, 21–23): If a small target protein can be immobilized and rigidly bound onto a larger symmetric assembly, EM particle images can be more readily aligned and classified, and structures too small to analyze alone could then become amenable to structure determination. Yeates et al. demonstrated the potential of this approach by fusing a designed ankyrin repeat protein (DARPin) (24, 25) to the outside of a previously designed protein nanocage (1); the bound green fluorescent protein (GFP) target was resolved at 3.8 Å resolution with only a single α-helical fusion anchoring the DARPin.