A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

Abstract

Myriad biological functions require protein-protein interactions (PPIs), and engineered PPIs are crucial for applications ranging from drug design to synthetic cell circuits. Understanding and engineering specificity in PPIs is particularly challenging as subtle sequence changes can drastically alter specificity. Coiled-coils are small protein domains that have long served as a simple model for studying the sequence-determinants of specificity and have been used as modular building blocks to build large protein nanostructures and synthetic circuits. Despite their simple rules and long-time use, building large sets of well-behaved orthogonal pairs that can be used together is still challenging because predictions are often inaccurate, and, as the library size increases, it becomes difficult to test predictions at scale. To address these problems, we first developed a method called the Next-Generation Bacterial Two-Hybrid (NGB2H), which combines gene synthesis, a bacterial two-hybrid assay, and a high-throughput next-generation sequencing readout, allowing rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way. After validating the NGB2H system on previously characterized libraries, we designed, built, and tested large sets of orthogonal synthetic coiled-coils. In an iterative set of experiments, we assayed more than 8,000 PPIs, used the dataset to train a novel linear model-based coiled-coil scoring algorithm, and then characterized nearly 18,000 interactions to identify the largest set of orthogonal PPIs to date with twenty-two on-target interactions.

Coiled Coils as Versatile Modules for Mammalian Cell Regulation

“…CCs have a small genetic footprint (about 100 base pairs per interaction module in most useful set comprising 4 heptads) which are subtle and typically expressed well, soluble and have favorable folding properties. The largest set of orthogonal CCs measures around 10-20 pairs [20,52]. This is substantially less than nucleic acid-based complementarity but is sufficient for most applications, and may potentially also be used for cellular barcoding.…”

Preorganized cyclic modules facilitate the self-assembly of protein nanostructures

“…Among the polypeptide modules that can facilitate the assembly of novel complexes, coiled-coils (CC) stand out as they can be designed de novo and nely tuned, as the rules underlying the specicity of the CC-forming peptides are wellunderstood and many orthogonal CCs are already available. [1][2][3][4][5][6] CCs are super-secondary structural elements composed of two or more a-helices intertwined in a supercoiled le-handed helix. Importantly, they display high specicity and known rules governing their oligomerization, enabling rational design.…”

Long-read sequencing to detect full-length protein-protein interactions

“…Methods for protein interaction mapping that utilize the cost and speed benefits from nextgeneration sequencing (NGS) are limited (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). In the case of two-hybrid systems, this is primarily because the information about which 'bait' and 'prey' interact are often on separate plasmids that are not physically connected.…”