Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

Abstract

A goal of de novo protein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric protein assemblies using these structures. We first connect designed helical repeat proteins (DHRs) to designed helical bundle proteins (HBs) to generate a large library of heterodimeric and homooligomeric building blocks; the latter have cyclic symmetries ranging from C2 to C6. All of the building blocks have repeat proteins with accessible termini, which we take advantage of in a second round of architecture guided rigid helical fusion (WORMS) to generate larger symmetric assemblies including C3 and C5 cyclic and D2 dihedral rings, a tetrahedral cage, and a 120 subunit icosahedral cage. Characterization of the structures by small angle x-ray scattering, x-ray crystallography, and cryo-electron microscopy demonstrates that the hierarchical design approach can accurately and robustly generate a wide range of macromolecular assemblies; with a diameter of 43nm, the icosahedral nanocage is the largest structurally validated designed cage to date. The computational methods and building block sets described here provide a very general route to new de novo designed symmetric protein nanomaterials.

De Novo Protein Design Using the Blueprint Builder in Rosetta

“…Designing de novo proteins using Rosetta has successfully provided many robust proteins with diverse topologies for various protein engineering purposes, including small molecule binding (Dou et al., 2018; Tinberg et al., 2013), therapeutic developments (Cao et al., 2020; Fleishman et al., 2011; Silva et al., 2019), orthogonal biological signaling system construction (Chen et al., 2019; Langan et al., 2019; Quijano‐Rubio et al., 2020), and material formation (Hsia et al., 2020; Pyles, Zhang, De Yoreo, & Baker, 2019; Ueda et al., 2020). A number of de novo proteins designed for the abovementioned applications have significantly different secondary structure features, and were generated using a similar pipeline, which all involved the key step of using the structure modifier in Rosetta, the blueprint builder (Huang et al., 2011; Marcos et al., 2018).…”

Designed proteins assemble antibodies into modular nanocages

“…To design proteins that assemble into nanocages, cyclically symmetric protein building blocks must be brought together to design larger nanocage architectures. Two common strategies for designing protein nanocages are computationally docking (17,18) or fusing (19)(20)(21) cyclically symmetric proteins (Fig. 1.…”

“…After fusion, WORMS checks that the new orientation of the oligomeric interfaces matches the desired geometry. This approach was successfully used to design dihedral and icosahedral nanocages, with some designs incorporating functional ankyrin loop-containing domains (19,20). WORMS is an excellent method for designing antibody nanocages by fusing alpha helical domains of antibody-binding proteins to existing cyclic oligomers in the orientations required to form nanocages, as this approach wouldn't require designing (i.e., mutating) any part of the antibody.…”

“…The WORMS computational protocol was used to rapidly sample all possible fusions from our building block library to identify those with the net rigid body transforms required to generate dihedral, tetrahedral, octahedral, and icosahedral AbCs (19,20).…”

Repeat proteins: designing new shapes and functions for solenoid folds

“…WORMS [23], instead, is a general method to create helix fusions that has been embedded in a design pipeline for symmetric architectures. The desired symmetry is provided by the user and the protocol looks for compatible terminal helices available for fusion.…”