Polymorphic Structures of an Artificial Nucleocapsid Evolved from a Circularly Permuted Bacterial Protein Cage

Abstract

Molecular self-assembly provides a route to nanoscale structures with defined sizes and shapes. Protein cages assembled from repeated subunits form compartments that can package molecular cargoes. Although amino acid substitutions can alter intersubunit interactions and cage architecture, how protein backbone connectivity relates to the cage architectures accessible during laboratory evolution remains unclear. Here we evolved an artificial nucleocapsid from a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS). Laboratory evolution increased the yield of purified nucleocapsids relative to the progenitor, and the evolved variant, NC119-3, packaged its encoding mRNA. Cryo-electron microscopy revealed a heterogeneous population of cages, within which we resolved 180- and 210-subunit cage families composed of 36 and 42 pentamers, respectively. Within each resolved family, particles with the same subunit stoichiometry differed in the arrangement and orientation of their pentameric building blocks. A 2.90 [A] reconstruction of a tetrahedral 180-subunit cage further revealed that sequence-identical subunits adopt distinct conformations depending on their positions within the assembly. These pentamer-based architectures differ from the domain-swapped T = 4 cage previously reported for nucleocapsids evolved from an AaLS variant with a different circular permutation site. Together with previous work, these findings show that RNA-packaging nucleocapsids evolved from scaffolds with different backbone connectivity can adopt distinct cage architectures.

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BibTeX RIS

BibTeX

@misc{li2026polymorphic,
  title = {{Polymorphic Structures of an Artificial Nucleocapsid Evolved from a Circularly Permuted Bacterial Protein Cage}},
  author = {H. Li and Łukasz Koziej and O. Kikko and K. Ishihara and T. Kagawa and K. Tajima and H. Shigematsu and C. Gerle and T. Matsuura and T. Numata and Y. Azuma and N. Terasaka},
  howpublished = {bioRxiv},
  year = {2026},
  month = oct,
  doi = {10.64898/2026.10.23.756749},
  url = {https://www.biorxiv.org/content/10.64898/2026.10.23.756749v1},
  note = {CC BY-NC-ND},
}

RIS

TY  - UNPB
TI  - Polymorphic Structures of an Artificial Nucleocapsid Evolved from a Circularly Permuted Bacterial Protein Cage
AU  - H. Li
AU  - Łukasz Koziej
AU  - O. Kikko
AU  - K. Ishihara
AU  - T. Kagawa
AU  - K. Tajima
AU  - H. Shigematsu
AU  - C. Gerle
AU  - T. Matsuura
AU  - T. Numata
AU  - Y. Azuma
AU  - N. Terasaka
PY  - 2026
DA  - 2026/10/23
PB  - bioRxiv
DO  - 10.64898/2026.10.23.756749
UR  - https://www.biorxiv.org/content/10.64898/2026.10.23.756749v1
AB  - Molecular self-assembly provides a route to nanoscale structures with defined sizes and shapes. Protein cages assembled from repeated subunits form compartments that can package molecular cargoes. Although amino acid substitutions can alter intersubunit interactions and cage architecture, how protein backbone connectivity relates to the cage architectures accessible during laboratory evolution remains unclear. Here we evolved an artificial nucleocapsid from a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS). Laboratory evolution increased the yield of purified nucleocapsids relative to the progenitor, and the evolved variant, NC119-3, packaged its encoding mRNA. Cryo-electron microscopy revealed a heterogeneous population of cages, within which we resolved 180- and 210-subunit cage families composed of 36 and 42 pentamers, respectively. Within each resolved family, particles with the same subunit stoichiometry differed in the arrangement and orientation of their pentameric building blocks. A 2.90 [A] reconstruction of a tetrahedral 180-subunit cage further revealed that sequence-identical subunits adopt distinct conformations depending on their positions within the assembly. These pentamer-based architectures differ from the domain-swapped T = 4 cage previously reported for nucleocapsids evolved from an AaLS variant with a different circular permutation site. Together with previous work, these findings show that RNA-packaging nucleocapsids evolved from scaffolds with different backbone connectivity can adopt distinct cage architectures.
ER  -

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