Beyond Dimerization: Harnessing Tetrameric Coiled-Coils for Nanostructure Assembly

Until now, scientists have used complementary dimerization modules of coiled coils to design modular protein structures, which they have called protein origami. Researchers from the Department of Synthetic Biology and Immunology have expanded the toolkit for modular nanostructure building blocks by using tetramerization modules instead. The dimeric building blocks were based on pairs of coiled coils, while the tetrameric building blocks consist of four helices that can orient parallel or antiparallel and bundle together.

It turned out that tetrameric modules significantly improve resistance to denaturation at the air-water interface, a common issue in cryo-electron microscopy. This new approach allowed the researchers to successfully determine the first three-dimensional structure of a designed protein made from coiled-coil bundles using cryo-electron microscopy.

In the study, they used different bundles of four helices: natural, designed; parallel, and antiparallel. This enabled them to design different topologies and assemble structures from two identical, but half-length, polypeptide chains. Experimental methods confirmed that the modules, according to the design, spontaneously formed a tetrahedral cage at the nanometer scale. At the same time, they demonstrated exceptional protein stability and their ability to refold after denaturation. Molecular dynamics analysis confirmed that the tetrahedral structure is stable, with the four-helix bundles contributing the most to the overall structural stability.

This research represents an important advance in the design of modular nanostructures based on polypeptides, which not only enhance stability and topological diversity but also enable the successful use of cryo-EM for structural analysis. The approach holds interesting potential for various applications, such as delivery systems. The authors of the paper are current and former members of the Department of Synthetic Biology and Immunology at the National Institute of Chemistry (Sara Vidmar, Tamara Šmidlehner, Jana Aupič, Žiga Strmšek, Ajasja Ljubetič, Roman Jerala), Soochow University (China), the Human Technopole Institute (Italy), and the Max Planck Institute (Germany). The article was published in Angewandte Chemie, International Edition.