Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X 10 C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-⌿-C, where ⌿ represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains. PDZ1 domains are 80 -90-amino acid modules present in numerous eukaryotic proteins. They were first described as a series of three internal, repeated elements within the postsynaptic density (PSD)-95 protein (1). In fact, the name PDZ is derived from three proteins first recognized to contain repeats of this domain: PSD-95; the Drosophila discs-large tumor suppressor protein, Dlg; and the mammalian tight-junction protein zona occludens-1, ZO-1 (2-5). PDZ domains have since been identified in a large number of multifunctional proteins, many of which are associated with specialized regions of cell to cell contact such as tight junctions, septate junctions, and synaptic junctions (6). The PDZ domain may be an evolutionarily old domain, as it has been detected in mammalian, nematode, yeast, plant, and bacterial genomic sequences by computer analysis (7).PDZ domains mediate protein-protein interactions by at least two distinct mechanisms. Certain PDZ domains bind directly to specific recognition sequences at the C terminus of transmembrane proteins. For example, the second PDZ domain of PSD-95 interacts with an S/T-X-V-COOH motif in N-methyl-D-aspartate receptor 2B subunits (8, 9) and in Shaker-type potassium channels (10). PDZ domains can also form heterotypic dimers with other PDZ domains. For instance, the Nterminal region of nNOS, which itself contains a PDZ domain, binds directly to PDZ domains in both PSD-95 and ␣ 1 -syntrophin, a component of the dystrophin-associated protein complex (DAPC) (11). A third possible mode of interaction, in which the consensus binding sequence is locat...

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