Analysis of PDZ Domain-Ligand Interactions Using Carboxyl-terminal Phage Display

• Published in: The Journal of biological chemistry Vol. 275; no. 28; pp. 21486 - 21491
• Main Authors: Fuh, Germaine, Pisabarro, M.Teresa, Li, Ying, Quan, Clifford, Lasky, Laurence A., Sidhu, Sachdev S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14-07-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacteriophage M13; Binding Sites; Capsid - chemistry; Capsid Proteins; Conserved Sequence; Humans; Kinetics; Ligands; Membrane Proteins - chemistry; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Tissue Proteins - chemistry; Peptide Library; Protein Conformation; Recombinant Proteins - chemistry; Sequence Alignment; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.28.21486

PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.