A map of WW domain family interactions

Abstract

WW domains are protein modules that bind proline-rich ligands. WW domain-ligand complexes are of importance as they have been implicated in several human diseases such as muscular dystrophy, cancer, hypertension, Alzheimer's, and Huntington's diseases. We report the results of a protein array aimed at mapping all the human WW domain protein-protein interactions. Our biochemical approach integrates parallel synthesis of peptides, protein expression, and high-throughput screening methodology combined with tools of bioinformatics. The results suggest that the majority of the bioinformatically predicted WW peptide ligands and most WW domains are functional, and that only about 10% of the measured domain-ligand interactions are positive. The analysis of the WW domain protein arrays also underscores the importance of the amino acid residues surrounding the WW ligand core motifs for specific binding to WW domains. In addition, the methodology presented here allows for the rapid elucidation of WW domain-ligand interactions with multiple applications including prediction of exact WW ligand binding sites, which can be applied to the mapping of other protein signaling domain families. Such information can be applied to the generation of protein interaction networks and identification of potential drug targets. To our knowledge, this report describes the first protein-protein interaction map of a domain in the human proteome.

Common Mechanism of Ligand Recognition by Group II/III WW Domains

“…The expression plasmid for GST fusion protein of the first WW domain of mouse FBP11 (FBP11A) was kindly provided by Dr. M. T. Bedford (26). The WW domains of HYPB, WAC, and Fe65 were inserted into pGEX-4T-2 vectors as described earlier by Hu et al (23).…”

“…Peptidestreptavidin/alkaline phosphatase reaction was used to monitor the relative strength of binding. Quality controls and checks were employed as previously described (23).…”

“…Their molecular masses were verified by MALDI-TOF mass spectrometry on a Voyager mass spectrometer (Applied Biosystems). The samples used in the proteomic mapping were prepared as described (23).…”

“…1 We also analyzed binding data generated by proteomic map-ping of the WW domain (23). We found that the WW domains previously classified as Groups II and III showed very similar ligand preference and that they were able to bind each other's cognate ligands.…”

Y65C Missense Mutation in the WW Domain of the Golabi-Ito-Hall Syndrome Protein PQBP1 Affects Its Binding Activity and Deregulates Pre-mRNA Splicing

“…In addition, we included 59 proline-rich peptides that did not contain the PPXY motif but scored positively in the proteomic map of the WW domain completed by the AxCell Co. (32). The total of 1958 peptides was synthesized on cellulose membranes using the SPOT technique (33) and probed with radioactively labeled GST-PQBP1-WW-WT or GST-PQBP1-WW-Y65C mutant (the list of the arrayed peptides is in supplemental Fig.…”