DoMY-Seq: A yeast two-hybrid-based technique for precision mapping of protein-protein interaction motifs.

Interactions between proteins are fundamental for every biological process and especially important in cell signaling pathways. Biochemical techniques that evaluate these protein-protein interactions (PPIs), such as in vitro pull downs and coimmunoprecipitations, have become popular in most laboratories and are essential to identify and validate novel protein binding partners. Most PPIs occur through small domains or motifs, which are challenging and laborious to map by using standard biochemical approaches because they generally require the cloning of several truncation mutants. Moreover, these classical methodologies provide limited resolution of the interacting interface. Here, we describe the development of an alternative technique to overcome these limitations termed "Protein Domain mapping using Yeast 2 Hybrid-Next Generation Sequencing" (DoMY-Seq), which leverages both yeast two-hybrid and next-generation sequencing techniques. In brief, our approach involves creating a library of fragments derived from an open reading frame of interest and enriching for the interacting fragments using a yeast two-hybrid reporter system. Next-generation sequencing is then subsequently employed to read and map the sequence of the interacting fragment, yielding a high-resolution plot of the binding interface. We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein-interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2. Thus, DoMY-Seq provides an unbiased alternative method to rapidly identify the domains involved in PPIs by advancing the use of yeast two-hybrid technology.

Non-full-length Water-Soluble CXCR4QTY and CCR5QTY Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors.

It was posited that functionalities of GPCRs require full-length sequences that are negated by residue deletions. Here we report that significantly truncated nfCCR5QTY and nfCXCR4QTY still bind native ligands. Receptor-ligand interactions were discovered from yeast 2-hybrid screening and confirmed by mating selection. Two nfCCR5QTY (SZ218a, SZ190b) and two nfCXCR4QTY (SZ158a, SZ146a) were expressed in E. coli. Synthesized receptors exhibited α-helical structures and bound respective ligands with reduced affinities. SZ190b and SZ158a were reconverted into non-QTY forms and expressed in HEK293T cells. Reconverted receptors localized on cell membranes and functioned as negative regulators for ligand-induced signaling when co-expressed with full-length receptors. CCR5-SZ190b individually can perform signaling at a reduced level with higher ligand concentration. Our findings provide insight into essential structural components for CCR5 and CXCR4 functionality, while raising the possibility that non-full-length receptors may be resulted from alternative splicing and that pseudo-genes in genomes may be present and functional in living organisms.