ABSTRACT
In an effort to develop a method for the high-throughput analysis of protein interaction interfaces, we devised a novel yeast genetic screening method, termed the "one- plus two-hybrid system," which efficiently selects specific missense mutations that disrupt known protein-protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid screening system is used first to positively select intact prey proteins, harboring informative missense mutations, from a large library of randomly generated mutant alleles. Next, among the isolated missense mutants of the prey proteins, interaction-defective mutants for a given protein (bait) are selected using the two-hybrid screening system. As a validation of the feasibility of this method, we utilized this technique to rapidly characterize the molecular determinants of the interactions between vitamin D receptor and its transcriptional coactivator protein, thyroid hormone receptor-associated protein 220. This efficient and rapid method should prove useful in the systematic analysis of large numbers of interaction interfaces.
Subject(s)
Alleles , Mutation, Missense/genetics , Proteins/genetics , Proteins/metabolism , Two-Hybrid System Techniques , Amino Acid Sequence , Base Sequence , Genes, Reporter/genetics , Genetic Vectors/genetics , Humans , Mutagenesis , Plasmids/genetics , Polymerase Chain ReactionABSTRACT
ASC-2 (activating signal co-integrator-2) is a transcriptional co-activator that mediates the transactivation of NRs (nuclear receptors) via direct interactions with these receptors. ASC-2 contains two separate NR-interaction domains harbouring a core signature motif, LXXLL (where X is any amino acid), named the NR box. Although the first NR box (NR box-1) of ASC-2 interacts with many different NRs, the second NR box (NR box-2) specifically interacts with only LXR (liver X receptor), whose transactivation in vivo requires heterodimerization with RXR (retinoid X receptor). Interestingly, RXR has been shown to enhance the LXR transactivation, even in the absence of LXR ligand via a unique mechanism of allosteric regulation. In the present study we demonstrate that LXR binding to an ASC-2 fragment containing NR box-2 (Co4aN) is enhanced by RXR and even further by liganded RXR. We also identified specific residues in Co4aN involved in its interaction with LXR that were also required for the ASC-2-mediated transactivation of LXR in mammalian cells. Using these mutants, we found that the Co4aN-LXR interaction surface is not altered by the presence of RXR and RXR ligand and that the Ser(1490) residue is the critical determinant for the LXR-specific interaction of Co4aN. Notably the NR box-2, but not the NR box-1, is essential for ASC-2-mediated transactivation of LXR in vivo and for the interaction between LXR-RXR and ASC-2 in vitro. These results indicate that RXR does not interact directly with NR box-1 of ASC-2, but functions as an allosteric activator of LXR binding to NR box-2 of ASC-2.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , DNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Ribonucleoproteins/metabolism , Allosteric Regulation , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Binding Sites , DNA-Binding Proteins/chemistry , Dimerization , Humans , Liver X Receptors , Mutagenesis, Site-Directed , Orphan Nuclear Receptors , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Plasmids , Protein Binding , Receptors, Cytoplasmic and Nuclear/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Transcriptional ActivationABSTRACT
To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.
