Design of Four-Component Protein Nanocages through Programmed Symmetry Breaking

Academic Background

Protein nanocages are highly symmetric protein assemblies widely used in vaccine development, drug delivery, and nanomaterial design. In nature, viruses often construct complex structures through symmetry breaking, particularly in high triangulation number (T) icosahedral structures. However, naturally occurring high-T tetrahedral or octahedral structures achieved through symmetry breaking have not been observed. To explore this field, researchers have proposed a general design strategy to construct high-T tetrahedral, octahedral, and icosahedral nanocages by pseudosymmetrizing trimeric building blocks.

Source of the Paper

The paper, authored by Sangmin Lee, Ryan D. Kibler, Green Ahn, Yang Hsia, Andrew J. Borst, Annika Philomin, Madison A. Kennedy, Buwei Huang, Barry Stoddard, and David Baker, was published in Nature. The authors are affiliated with the Department of Biochemistry at the University of Washington, the Institute for Protein Design, the Howard Hughes Medical Institute, Pohang University of Science and Technology (POSTECH), and the Fred Hutchinson Cancer Research Center. The paper was published online on July 11, 2024.

Research Process

1. Design Strategy

The researchers proposed a hierarchical design strategy to construct high-T tetrahedral, octahedral, and icosahedral nanocages by pseudosymmetrizing trimeric building blocks. The specific steps are as follows: 1. Design of T=1 Cages: First, C3 symmetric homotrimers were designed and arranged along the symmetry axes of tetrahedral, octahedral, and icosahedral architectures to form T=1 nanocages. 2. Extraction of Cyclic Substructures: Homotrimers were replaced with pseudosymmetric heterotrimers to extract cyclic substructures (crowns). These heterotrimers consist of three distinct chains and lack one trimer-trimer interface, isolating the substructures from the cage. 3. Construction of T=4 Cages: The extracted crowns were combined with new homotrimers, arranged along the symmetry axes of tetrahedral, octahedral, and icosahedral architectures, to form T=4 nanocages.

2. Design of Pseudosymmetric Heterotrimers

The researchers employed an interface transplantation approach, grafting interfaces from different homotrimers onto a host trimer to construct pseudosymmetric heterotrimers. Experimental validation confirmed that these heterotrimers formed the expected structures and had varying arm lengths, facilitating subsequent assembly.

3. Design and Validation of Nanocages

The researchers designed T=1 tetrahedral, octahedral, and icosahedral nanocages and experimentally validated their structures. Subsequently, crowns were extracted and combined with new homotrimers to construct T=4 nanocages. The structures of these nanocages were verified using negative-stain electron microscopy (NSEM) and cryo-electron microscopy (cryo-EM).

Key Results

1. Structure of T=4 Nanocages: The researchers successfully constructed T=4 tetrahedral, octahedral, and icosahedral nanocages, composed of 48, 96, and 240 subunits, respectively, with diameters of 33 nm, 43 nm, and 75 nm. These nanocages feature four distinct chains and six different protein-protein interfaces.
2. Validation of Pseudosymmetric Heterotrimers: Experimental validation confirmed that pseudosymmetric heterotrimers formed the expected structures and had varying arm lengths, facilitating subsequent assembly.
3. Thermal Stability and pH Tolerance of Nanocages: Experiments demonstrated that T=4 octahedral and icosahedral nanocages remained stable below 70°C and maintained structural integrity above pH 5.3, indicating their potential as delivery vehicles.
4. Cellular Internalization of Nanocages: By fusing one chain of the icosahedral nanocage to an asialoglycoprotein receptor (ASGPR) binding protein, the researchers successfully achieved internalization of the nanocages in liver cells.

Conclusion

The study presents a general design strategy for constructing high-T tetrahedral, octahedral, and icosahedral nanocages by pseudosymmetrizing trimeric building blocks. These nanocages, with their complex structures and multiple functionalization sites, provide a new platform for vaccine development and drug delivery. Notably, the T=4 icosahedral nanocage offers a large internal volume for packaging nucleic acids and other macromolecules.

Research Highlights

1. Innovative Design Strategy: The study introduces a novel design strategy for constructing high-T tetrahedral, octahedral, and icosahedral nanocages through pseudosymmetrization.
2. Complex Nanocage Structures: Successful construction of T=4 nanocages with four distinct chains and six different interfaces demonstrates their potential in vaccine development and drug delivery.
3. Experimental Validation: The structures of the nanocages were verified using NSEM and cryo-EM, and their thermal stability and pH tolerance were experimentally confirmed.
4. Cellular Internalization Experiments: Successful internalization of nanocages in liver cells highlights their potential as delivery vehicles.

Research Significance

The study provides new insights into the design of protein nanocages, demonstrating the feasibility of constructing complex nanostructures through pseudosymmetrization. These nanocages have broad applications in vaccine development, drug delivery, and nanomaterial design. In particular, the T=4 icosahedral nanocage, with its large internal volume, offers new tools for gene therapy and vaccine development.