Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA)² (2–6), where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7, 8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction “hub” according to their binding affinity.Quantitative estimation of protein-protein interactions in vivo will require the amount of the complex to be directly measured or the level of reconstituted reporters to be directly proportional to the strength of the interactions. To achieve quantitative analysis of protein interactions in eukaryotic expression system, we have designed a yeast surface two-hybrid (YS2H) system that can express a pair of proteins, one protein as a fusion to a yeast cell wall protein, agglutinin, and the other in a secretory form. When two proteins interact in this system, they associate in the secretory pathway, and the prey that would otherwise be released into the media is captured on the surface by the bait. We have devised two different schemes to quantitatively estimate the affinity of two interacting molecules: flow cytometric detection of antibody binding to the epitope tags fused to the prey and the bait, and the GFP readout from the complementation of split GFP fragments fused to the prey and the bait. They are induced under a bi-directional promoter to promote a synchronized and comparable level of expression.Herein we demonstrate the quantitative nature of YS2H in predicting the affinity between two interacting proteins, particularly in the range of 100 pm to 10 μm. This feature allowed us to examine specific interactions between antigen and antibody, to identify hot spots of allosteric activation in integrins, and to isolate camelid heavy chain-only antibodies against botulinum neurotoxin as components of therapeutic agents to treat botulism (9). With the incorporation of PCA technique into the YS2H, our system may be developed into an in vivo tool to measure the kinetics of protein-protein interactions. Potential applications of YS2H include affinity maturation of antibodies, differentiation of weak to high affinity binders to the hub protein in interaction networks, and confirmation of hypothetical interacting pairs of proteins in a high throughput manner.