Cyclic Peptides as Non-carboxyl-terminal Ligands of Syntrophin PDZ Domains

Abstract

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...

Energetic Determinants of Internal Motif Recognition by PDZ Domains

“…These findings are consistent with previous studies in which it was found that the syntrophin PDZ domain could recognize internal motifs present in peptides cyclized by an internal disulfide linkage (24). Recognition in this case was also dependent on proper structural presentation, as reduction of the cyclic disulfide linkage eliminated binding.…”

Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5

“…The wells were then incubated for 2 h at room temperature with 10 10 recombinant phage particles. After extensive washing with phosphate-buffered saline, pH 7.4, and 0.1% Tween 20, the amount of retained phage peptide was quantitated in a phage ELISA using an anti-M13 antibody (53).…”

Interaction of γ1-Syntrophin with Diacylglycerol Kinase-ζ

“…All peptides contained an additional 4-amino acid linker (SGSG) at the N terminus and an N-terminal biotin. Overlay assays were carried out as described previously (8,28) with the following modifications. Signals generated by enhanced chemiluminescence were captured with a Digital Image Station (Kodak).…”

“…Most known PDZ domain-mediated interactions occur by the recognition of short C-terminal peptide motifs and require that the peptide ligands have a free carboxylate group (29), but in some cases internal peptide motifs that are well removed from the C terminus can mediate interactions with PDZ domains (12,28). Therefore, we tested whether the addition of a C-terminal FLAG tag to DGK-would affect its interaction with ␥1-syntrophin.…”