Molecular machines based on coiled-coil protein origami

Project Information

MaCChines

Grant agreement ID: 787115

DOI

10.3030/787115

Project closed

EC signature date 31 August 2018

Start date 1 September 2018

End date 31 August 2024

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 497 125,00

EU contribution

€ 2 497 125,00

Coordinated by

KEMIJSKI INSTITUT
Slovenia

Periodic Reporting for period 4 - MaCChines (Molecular machines based on coiled-coil protein origami)

Reporting period: 2023-03-01 to 2024-08-31

Summary of the context and overall objectives of the project

Proteins are the most versatile biopolymers that underlay the functions and structures of all living organisms. Natural proteins form a wide range of different structures with a large variety of shapes and functions but represent only a tiny fraction of all possible polypeptide structures. The grand challenge is to design new proteins that have not evolved in nature and are based on alternative design principles and may have new interesting features that could be designed. Within the project MaCChines we aimed to investigate and explore the potentials of designed proteins and biological systems based on coiled-coil building modules (coiled-coil protein origami - CCPO) and take advantage of the unique properties of this design platform.
The general objective of MaCChines was to build and expand the foundations of a new branch of protein design science based on the modular design principles, to develop and exploit unique features of de novo designed coiled-coil protein origami (CCPO), to enable new drug delivery technologies, construction of complex nano-scaffolds, new materials, sensors, rational design of molecular machines and new advanced therapeutic modalities, for potential translation to the clinic, which we are pursuing through the established Center for Technologies of Gene and Cell therapy (CTGCT) at the National institute of chemistry.

More specific objectives investigated in the action were:
1. functionalize CCPO cages by the addition of functional protein domains
2. new types of regulation of CCPO assembly and disassembly, mediated by diverse chemical and biological signals
3. knotting topology into the self-assembling designed structures
4. Extended repertoire of building modules beyond CC dimers
5. Apply the developed modules and tools for new types of regulation into biological systems

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Within the project, we achieved important advances in several exciting directions along the proposed directions of the ERC AdG MaCChines project including some new ideas that have been conceived and realized during this project, such as the design of the folding pathways.
We have developed several types of functionalized CC modules and implemented them for different functions:
1. New metal and pH- dependent coiled coil modules (Aupič et al., Chem-Bio.Chem. 2018, Aupič et al., Sci.Adv. 2022).
2. Application of CC modules for modulation of localization and regulation of biochemical/ biological processes (Lebar et al., Nat.Chem.Biol. 2020).
3. Fusion of CC modules with split proteases to generate fast responsive cellular logic (Fink et al., NatChemBiol 2019).
4. Use of CC dimeric modules for the assembly of liquid protein condensates (designed liquid-liquid phase separation) in mammalian cells (Ramšak et al., Nat.Comm. 2023).
5. Structure determination of CCPO cages and confirmation of knotted structure (Satler et al, JACS 2024). The structure validates the modular CC-based protein design strategy, and a recent cryoEM determination of the tetrahedron that contained a four-helix coiled coil confirmed the 3D topology according to the design (Vidmar et al., AngewChemie accepted 2024).

We introduced several new strategies for CCPO assembly that mediated the construction of new protein folds and assemblies:
a) Multichain CCPO assembly (Lapenta et al., Nat.Comm. 2021). Self-assembly of CC-based nanostructures from several chains facilitates the design of more complex assemblies and the introduction of functionalities. This could facilitate the construction of dynamic multi-chain CC-based complexes.
b) Design of the designed protein folding pathway and use of multiple copies of the same type of the CC peptide (Aupič et al., Nat.Comm. 2021). Investigation of the folding pathway of a CCPO tetrahedral cage revealed that CCPO folding is dominated by the intra-chain distance between CC modules in the primary sequence and folding intermediates.
c) Multichain design of protein cages facilitated by intein-mediated cyclization and intramolecular interactions (Snoj et al., Chem.Sci. 2024). Preorganized subunits comprised cyclization through spontaneous self-splicing of split intein and intramolecular coiled-coil dimers t construct multichain assemblies.
d) Large rigid fibrils based on connecting spectrin repeat modules by designed CC dimers (Mezgec et al., ACS Nano, 2024). Extended rigid nanoscale filaments were based on spectrin repeats composed of spectrin repeats and connected via CC-heterodimers, which could be decorated with protein domains and regulated by metal ions.
We obtained and successfully finished an ERC PoC project CCedit, where we used the designed CC modules to improve the efficiency of genome editing and applied for IP protection.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The ERC AdG MaCChines project achieved important advances in protein design and modular assembly, surpassing the state of the art in synthetic biology, biochemistry, and biomaterials science. Key innovations include functionalized coiled-coil (CC) modules for tunable protein folding, self-assembly, and cellular process regulation. Metal and pH-responsive CC modules enabled reversible interactions for dynamic assemblies, while orthogonal CC heterodimers, controlled gene transcription, localization, and CRISPR-dCas9 activity. Split protease-activated CC circuits implemented rapid Boolean logic in mammalian cells, and CC-driven liquid-liquid phase separation (LLPS) condensates revealed insights into membrane-less organelles.
The advances in CCPO in this project include streamlined folding pathways using identical modules, multichain assemblies with proteolytic switches, and cyclization-based subunit organization for complex structures. Extended nanoscale filaments combining spectrin repeats and CC dimers provided tunable scaffolds for biomaterials. A novel platform for modular allosteric protein regulation encoded logic gates in cells, allowing precise control of protein activity. These innovations culminated in practical applications, including genome editing optimization via the ERC PoC CCedit project. Together, the results establish a versatile framework for synthetic biology, with implications for medicine, biotechnology, and materials science.
Results generated within MaCChines introduced several innovative concepts in protein design that could apply also to the design of other programmable polymers such as nucleic acids.

The key project Contributions Beyond the State of the Art
• Novel Modular Designs: Introduced CC-based designs that regulate protein folding, self-assembly, and cellular functions in several innovative ways.
• Rapid and Dynamic Systems: Developed systems capable of fast responses (minutes) in living cells, advancing applications in synthetic biology and biomedicine.
• Scalable and Simplified Assembly: Created strategies for modular, scalable protein origami and biomaterial scaffolding, reducing complexity and enhancing functionality.
• Biochemical Innovation: Enabled new insights into LLPS and introduced a platform for dynamic regulation of biological processes.

These achievements position the project as a pioneer in modular protein engineering and synthetic biology, opening new avenues for research and application in biotechnology and medicine. The demonstration and concepts for the introduction of coiled coil-based modules into mammalian cells represent a foundation for their wider use by other researchers and for diverse applications.

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