Academic Background

Protein self-assembly is a ubiquitous phenomenon in biological systems, performing a wide range of functions from structural support to biochemical regulation. Despite significant progress in protein design in recent years, existing self-assembling protein structures typically rely on strict symmetry, which limits their size and complexity. To overcome this limitation, researchers drew inspiration from the pseudosymmetry observed in bacterial microcompartments and viral capsids, developing a hierarchical computational method to design large pseudosymmetric self-assembling protein nanomaterials. This study aims to break the constraints of strict symmetry and design larger, more complex protein nanocages, thereby expanding the diversity of self-assembling protein structures.

Source of the Paper

The research was conducted by a team including Quinton M. Dowling, Young-Jun Park, Chelsea N. Fries, and others from the University of Washington. The paper was published in Nature in 2024. The research team primarily comes from the Institute for Protein Design and the Department of Biochemistry at the University of Washington, with some researchers also affiliated with the Howard Hughes Medical Institute.

Research Process and Results

1. Design of Pseudosymmetric Heterotrimers

The research team began with a stable homotrimeric protein (PDB ID: 1wa3) from the hyperthermophilic bacterium Thermotoga maritima to design pseudosymmetric heterotrimers. By introducing mutations to disrupt the stability of the homotrimer and combining compensatory mutations to restore trimer assembly, the researchers successfully designed three different pseudosymmetric heterotrimers. These heterotrimers, through different amino acid sequence combinations, formed new protein-protein interfaces, thereby achieving pseudosymmetry.

Experimental Methods:

• Mutation Screening: The Rosetta software was used to calculate the effects of mutations on trimer stability, screening for mutations that could disrupt trimer stability and their compensatory mutations.
• Experimental Validation: The mutated trimers were expressed using the E. coli expression system, and their assembly capabilities were verified using Native PAGE and mass spectrometry.

Results:

The researchers successfully designed three pseudosymmetric heterotrimers and experimentally verified their assembly capabilities. These heterotrimers were able to bind with known pentamers to form nanocage structures.

2. Design of 240-Subunit Nanocages

Based on the designed pseudosymmetric heterotrimers, the researchers further designed a 240-subunit nanocage with icosahedral symmetry. By docking the heterotrimers with homotrimers and designing new protein-protein interfaces, the researchers successfully constructed the gi4-f7 nanocage.

Experimental Methods:

• Docking and Design: Computational docking methods were used to dock heterotrimers with homotrimers, and new interfaces were designed.
• Expression and Purification: The components of the nanocage were expressed using the E. coli expression system and purified using IMAC and SEC.
• Structural Validation: The nanocage structure was resolved using cryo-electron microscopy (cryo-EM).

Results:

The cryo-EM structure revealed that the gi4-f7 nanocage had a diameter of approximately 49 nm, closely matching the design model. The successful assembly of this nanocage validated the feasibility of the pseudosymmetric design approach.

3. Discovery and Design of 540-Subunit Nanocages

In the cryo-EM images of gi4-f7, the researchers unexpectedly discovered a larger 71 nm nanocage, named gi9-f7. This nanocage consisted of 540 subunits, with a structure similar to gi4-f7 but with larger size and more complex assembly.

Experimental Methods:

• Structural Resolution: The structure of gi9-f7 was resolved using cryo-EM.
• Assembly Validation: The assembly of gi9-f7 was optimized by adjusting the ratio of heterotrimers to homotrimers.

Results:

The cryo-EM structure showed that the gi9-f7 nanocage had a diameter of approximately 71 nm, closely matching the design model. The discovery of this nanocage further demonstrated the scalability of the pseudosymmetric design approach.

4. Design of Extensible Nanocages

Based on the designs of gi4-f7 and gi9-f7, the researchers proposed a strategy for designing extensible nanocages. By introducing a new homotrimer (bbb), the researchers successfully designed the gi16-f7 nanocage, which had a diameter of 96 nm and consisted of 960 subunits.

Experimental Methods:

• Structural Resolution: The structure of gi16-f7 was resolved using cryo-EM.
• Assembly Validation: The assembly of the nanocage was verified using dynamic light scattering (DLS) and cryo-EM.

Results:

The cryo-EM structure revealed that the gi16-f7 nanocage had a diameter of approximately 96 nm, closely matching the design model. The design of this nanocage further extended the size and complexity of pseudosymmetric nanocages.

Conclusions and Significance

Through a hierarchical pseudosymmetric design approach, this study successfully designed the largest computationally designed protein nanocages to date. The size and complexity of these nanocages far exceed previous designs, demonstrating the immense potential of the pseudosymmetric design approach in the field of protein self-assembly. This research not only expands the diversity of self-assembling protein structures but also provides new insights for future nanomaterial design.

Research Highlights

1. Pseudosymmetric Design: By breaking strict symmetry, larger and more complex protein nanocages were designed.
2. Hierarchical Design Strategy: By first designing pseudosymmetric heterotrimers and then using them as building blocks for larger nanocages, precise design of complex structures was achieved.
3. Cryo-EM Validation: The structures of the designed nanocages were resolved using cryo-EM, validating the accuracy of the design approach.
4. Scalability: By introducing new homotrimers, extensible nanocages were successfully designed, showcasing the broad application potential of this method.

Application Value

The designed protein nanocages have wide-ranging applications in drug delivery, enzyme encapsulation, and vaccine development. For example, the researchers successfully displayed the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein on the surface of the nanocages and verified their ability to activate B cells, demonstrating their potential application in vaccine development.

Summary

Through a hierarchical pseudosymmetric design approach, this study successfully designed the largest computationally designed protein nanocages to date, showcasing the immense potential of the pseudosymmetric design approach in the field of protein self-assembly. This research not only expands the diversity of self-assembling protein structures but also provides new insights for future nanomaterial design.