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

Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Coiled-coils have been used as a model to study the sequence determinants of specificity. However, building well-behaved sets of orthogonal pairs of coiled-coils remains challenging due to inaccurate predictions of orthogonality and difficulties in testing at scale. To address this, we develop the next-generation bacterial two-hybrid (NGB2H) method, which allows for the rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way using next-generation sequencing readout. We design, build, and test large sets of orthogonal synthetic coiled-coils, assayed over 8,000 PPIs, and used the dataset to train a more accurate coiled-coil scoring algorithm (iCipa). After characterizing nearly 18,000 new PPIs, we identify to the best of our knowledge the largest set of orthogonal coiled-coils to date, with fifteen on-target interactions. Our approach provides a powerful tool for the design of orthogonal PPIs.

Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays.

In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.

Directed network wiring identifies a key protein interaction in embryonic stem cell differentiation.

Cell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain ("pY-clamps") whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs.