A multiplexed bacterial two-hybrid for rapid characterization of protein–protein interactions and iterative protein design

Highlights:

• A method for simultaneously studying over 20,000 protein-protein interactions
• Computational method for designing orthogonal sets (where each peptide binds only to its partner)
• Measurement of over 26,000 interactions between peptides to form coiled coils
• New more accurate function to predict binding between coiled coils
• Designed the largest orthogonal coiled coil set known to date, with fifteen pairs!

The paper will enable the design of complex protein origami structures for drug delivery and other applications in synthetic biology.

Protein-protein interactions (PPIs) are key to biological functions and are used in applications ranging from drug design to synthetic cell circuits. In our research group, designed coiled-coil proteins have been used in many ways to assemble novel protein structures to regulate the function of human cells, including potential applications for therapeutics (Ljubetič et al. Nat.Biotech. 2017, Fink et al. Nat.Chem.Biol. 2019, Merljak et al., Nat.Commun. 2023, Praznik et al. Nat.Comm. 2019, Lebar et al. Nat.Chem.Biol. 2020, Aupič et al. Sci.Adv. 2022, Lapenta et al. Nat.Comm. 2021, Lainšček et al. Nat.Comm. 2022, Satler et al. JACS 2023).

An extremely important concept in the design of PPIs is orthogonality. In an orthogonal set, only the individual well-defined pairs interact with each other, and the other interactions are much weaker. Orthogonality thus simplifies further applications, as we can determine exactly which parts of the designed system interconnect with each other.

We have developed the largest orthogonal set of coiled coils (CCs) known to date, which is also larger than any known set from natural or engineered proteins. Coiled coils consist of two α-helices that connect via precisely defined amino acid residues. Until now, we had no way to design large orthogonal sets, so we developed a new computational method based on graph theory that allows for the rapid selection of orthogonal sets based on the calculated interactions. Also, there was no method available to predict the orthogonality of CCs. Therefore, we developed an experimental method (NGB2H - Next Generation Bacterial Two Hybrid) that allows the simultaneous measurement of many protein interactions. In a single tube, this method can measure more than 10,000 interactions at once.  In total, we have measured the interaction strength between more than 26,000 coiled coils. The largest orthogonal set we have developed has 15 pairs, which is twice as many as the existing orthogonal CC set (Crooks et al. Biochemistry, 2017).

Our approach provides an efficient tool for designing orthogonal PPIs, and the computational and experimental methods developed will be useful to a wide range of scientists.

The research was led by dr. Ajasja Ljubetič and preof. Roman Jerala, colleagues at the Department of Synthetic Biology and Immunology at the National Institute of Chemistry. The research was carried out in collaboration with Sri Kosuri’s laboratory at the University of California, LA, USA, where they made key contributions to the experimental part.

The results were published in Nature Communications (IF = 17.7).

Link to article: https://www.nature.com/articles/s41467-023-38697-x

Contact for more information: ajasja.ljubetic(at)ki.si, roman.jerala(at)ki.si