Design of multi-scale protein complexes by hierarchical building block fusion

Abstract

A systematic and robust approach to generating complex protein nanomaterials would have broad utility. We develop a hierarchical approach to designing multi-component protein assemblies from two classes of modular building blocks: designed helical repeat proteins (DHRs) and helical bundle oligomers (HBs). We first rigidly fuse DHRs to HBs to generate a large library of oligomeric building blocks. We then generate assemblies with cyclic, dihedral, and point group symmetries from these building blocks using architecture guided rigid helical fusion with new software named WORMS. X-ray crystallography and cryo-electron microscopy characterization show that the hierarchical design approach can accurately generate a wide range of assemblies, including a 43 nm diameter icosahedral nanocage. The computational methods and building block sets described here provide a very general route to de novo designed protein nanomaterials.

Fig. 1. Overview of the rigid hierarchical fusion approach.

a Hetero- (yellow/green) and homo- (red) oligomeric helical bundles are fused to de novo helical repeat proteins (shades of blue) (left) to create a wide range of building blocks using HelixDock and HelixFuse (center). Symmetric units shown in gray. b Twenty representative HelixFuse outputs overlaid in groups of five display the wide range of diversity that can be generated by using a single helical bundle core (symmetric units hidden for clarity). c Building blocks are further assembled into higher-ordered structures through helical fusion (WORMS). The examples are cyclic crowns (top), dihedral rings (middle), and icosahedral nanocages (bottom); additional details available in their respective sections.

Fig. 2. Homo-oligomer diversification by repeat protein fusion.

One central oligomer unit is shown in red and fused DHR in blue while other symmetrical units in grey. HelixDock (HD); HelixFuse (HF). a C3_HD-1069, with designed loop shown in yellow, b C3_HF_Wm-0024A, with additional WORMS fusion shown in yellow), and c C3_nat_HF-0005. Overlay of the design model (purple/gray) and crystal structure (yellow/white) shows the overall match of the backbone. Inset shows the correct placement of the rotamers in the designed junction region. d C4_nat_HF-7900; design model (purple/grey) and cryo-EM map (yellow/white), with insert highlighting the the high resolution (~3.8 Å) density. e C5_HF-3921 as characterized by cryo-EM, with inset showing density surrounding the designed junction. f C5_HF-2101, g C5_HF-0019, h C6_HF-0075, and i C6_HF-0080 all showed a good overall match to its negative-stain EM 2D class averages (top) from one direction, using a predicted projection map (bottom).

Fig. 3. Design of cyclic crowns from heterodimeric building blocks.

a Hetero-dimeric HB (green/yellow) fused with different DHRs (shades of blue) were fused together using WORMS by enforcing a specific overall cyclic symmetry (C3 and C5 shown). b The backbones of the crystal structure (yellow/white) of C3_Crn-05 overlaid with the design model (purple/gray). Insets show the backbone matching focused at each of the fusion locations. c A C5 crown (C5_Crn-07, asymmetric unit in red) was fused to DHR units on either outward facing (“C5_Crn_HF-12”, blue arrow) or inward facing termini (“C5_Crn_HF-26”, dark blue arrow). The two structures were then merged together to generate a double fusion (“C5_Crn_HF-12_26”, black arrow). d Cryo-EM class average of the fused 12_26 structure; the major C5 species shown. 3D reconstruction shows the main features of the designed structure are present, as is also evident in the class average (right).

Fig. 4. Design of two-component dihedral rings.

a Two different homodimeric HBs (red) with DHR extensions (shades of blue) were aligned to their respective symmetrical axes with dihedral symmetry. An additional heterodimer (green/yellow) was placed between them through architecture aware helical fusion, generating an 8-chain D2 ring. b The final asymmetric unit shown in green/yellow while the inset preserves the original colors. c Negative-stain EM followed by 2D average and 3D reconstruction of D2_Wm-01 and D2_Wm-01_trunc show that the major features of the designs were recapitulated (left) designed model, (middle) overlay of the designed models with the 3D reconstructions, (right) 2D averages.

Fig. 5. Design of assemblies with point group symmetry.

a Tetrahedron design schematic. A HB and a C2 homo-oligomeric made from ankyrin repeat proteins were aligned to their respective tetrahedral symmetry axis (red), and connected via fusion to Ankyrin repeat monomers (blue) to generate the target architecture. b 3D reconstruction reveals a well fitting map of T_Wm-1606. c Icosahedral design schematic. Libraries of unverified cyclic fusion homo-dimers and trimers were aligned to the corresponding icosahedral symmetry axes. Using WORMS, fusions to DHRs split in the center that hold the two homo-oligomers in the orientations which generate icosahedral structures were identified. d Cryo-EM 3D reconstruction of I32_Wm-42 closely matches the designed model.