A High Efficiency Strategy for Binding Property Characterization of Peptide-binding Domains

Abstract

A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale. Molecular & Cellular Proteomics 5:1368 -1381, 2006.

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

“…The PDZ ligand library was constructed in our previous work [24] . In brief, the library was constructed mainly by collecting all the non-redundant ligand clones isolated from all the Y2H screenings of random peptide libraries with representative PDZ domains and all the predicted ligand clones constructed for confirmations.…”

“…However, they are all based on large-scale screenings for each bait domain, which are relatively labor-intensive and timeconsuming. In our previous work, for more efficiently characterizing the binding properties of peptide-binding domains, we developed a systematic strategy of highthroughput validation screening of a specialized candidate ligand library [24] . A PDZ ligand library for characterizing PDZ domain family has been constructed mainly by collecting all the non-redundant ligand clones isolated from Y2H screenings of random peptide libraries with representative PDZ domains and the predicted ligand clones constructed for confirmations [24] .…”

Systematic Analysis of a Simple Adaptor Protein PDZK1: Ligand Identification, Interaction and Functional Prediction of Complex

“…Because PDZK1 PDZ domains commonly mediate the formation of protein complexes at the membrane, the ligands found at the membrane were chosen for one-to-one Y2H confirmation. The GAL4 AD plasmids expressing the carboxyl-terminal 13 amino acid residues of the candidate ligands of PDZK1-PDZ1 were first constructed based on our previously described method [22]. Each constructed plasmid was co-transformed with PDZK1-PDZ1 bait plasmid into yeast strain CG1945.…”

“…The PDZ ligand library was the collection of all the non-redundant positive clones isolated from random peptide library screening by other PDZ domains previously [22]. The one-to-one Y2H method was used for validation screening of the current PDZ ligand library consisting of 242 non-redundant PDZ ligand clones.…”

“…A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening experiments, 1.4×10 7 clones were obtained for the random peptide library. The GAL4 BD-PDZ fusion bait plasmid was transformed into the yeast strain CG1945 using the lithium acetate protocol as previously described [22].…”