De novo design of quasisymmetric two-component protein cages | Nature
Subjects
- Molecular self-assembly
- Protein design
Abstract
Quasisymmetric icosahedral viral capsids achieve larger sizes than possible with strictly symmetric icosahedra by tessellating pentagons and hexagons using a single subunit that adopts different conformations in symmetrically non-equivalent locations1,2. Recapitulating such quasisymmetric architectures through computational design is a considerable challenge in nanomaterials engineering. Here we introduce a computational design strategy based on geometric frustration to generate two-component, quasisymmetric protein cages with customizable properties. We designed complementary trimeric and dimeric protein components that co-assemble into positively curved local hexagonal assemblies. Hexagonal lattices cannot tile spherical surfaces; instead, the components form closed sphere-like cage assemblies through incorporation of curvature-inducing pentagonal defects, as evidenced by electron microscopy. By designing dimers that encode different local curvatures, we programmed cage dimensions ranging from 40 to over 200 nm in diameter and with molecular weights from 2 MDa to over 50 MDa, comparable with natural virus capsids. We further functionalized these large cages with additional protein domains to enable ribonucleoprotein cargo loading and cellular uptake. Fluorescently labelled cage assemblies expressed in mammalian cells function as rheological probes and cargo recruiters, enabling a systematic study of size-dependent cytoplasmic diffusion and protein localization. Thus, the quasi-symmetry that has long fascinated structural biologists can now be achieved by computational protein design, with immediate applications to biologics delivery and molecular cell biology.
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Fig. 1: Computational design strategy for generating quasisymmetric particles.The alternative text for this image may have been generated using AI.
Fig. 2: Cage size can be programmed through the curvature of designed dimeric linkers.The alternative text for this image may have been generated using AI.
Fig. 3: Cryo-EM characterization of in vitro assembled cages.The alternative text for this image may have been generated using AI.
Fig. 4: Cage assembly in vitro and in living cells for cargo packaging and rheological probes.The alternative text for this image may have been generated using AI.
Data availability
The principal data supporting the findings of this work are available in the figures and the Supplementary Information. Design models for the cage building blocks are available via Zenodo at https://doi.org/10.5281/zenodo.18892841 (ref. 46). The heterodimer protein pair LHD101 was derived from ref. 19 (PDB ID 7MWR). Coordinates and structure factors are available in the PDB with the following accession codes: 9NDL (C2-B-α20), 9OM3 (T = 3 cage, cryo-EM structure) and 9OP9 (T = 3 cage, cryo-ET structure). Source data are provided with this paper.
Code availability
RFDiffusion code can be downloaded from https://github.com/RosettaCommons/RFdiffusion. ProteinMPNN is available from https://github.com/sokrypton/ColabDesign/. AlphaFold2 is available from https://github.com/google-deepmind/alphafold. Scripts for generating curved hexagon are available via Zenodo at https://doi.org/10.5281/zenodo.18892841 (ref. 46)
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