Cyclic peptides as non-carboxyl-terminal ligands of syntrophin PDZ domains.
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 X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi 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.
Department of Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
S H
SH
Gee
S A
SA
Sekely
C
C
Lombardo
A
A
Kurakin
S C
SC
Froehner
B K
BK
Kay
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000378:metabolism; D001665:Binding Sites; D015374:Biosensing Techniques; D002135:Calcium-Binding Proteins; D049029:Dystrophin-Associated Proteins; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism
D002135:Calcium-Binding Proteins; D049029:Dystrophin-Associated Proteins; D008024:Ligands; D008565:Membrane Proteins; D009124:Muscle Proteins; D019151:Peptide Library; D010456:Peptides, Cyclic; D011498:Protein Precursors; D015222:Sodium Channels; C091824:syntrophin; C091825:syntrophin alpha1; D004744:Enkephalin, Methionine; C036452:peptide F