title: Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.
abstract: PDZ domains are modular protein-protein interaction domains that bind to specific C-terminal sequences of membrane proteins and/or to other PDZ domains. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal peptide ligands and heterodimerize with the extended nNOS PDZ domain. The capacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate- binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K(+) channel Kv1.4. The same mutation affected nNOS binding to alpha1- and beta1-syntrophin PDZ domains to a lesser extent, due in part to the stabilizing effect of tertiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin that forms a tertiary contact with the nNOS PDZ domain. This residue is important for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basis, we have identified the C terminus of the Kir2.1 channel as a possible binding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.
authors.affiliation: Department of Cellular and Molecular Medicine, Neuromuscular Research Group, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5. stevegee@uottawa.ca
authors.forename: S H
authors.initials: SH
authors.lastname: Gee
authors.forename: S
authors.initials: S
authors.lastname: Quenneville
authors.forename: C R
authors.initials: CR
authors.lastname: Lombardo
authors.forename: J
authors.initials: J
authors.lastname: Chabot
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism*; D019281:Dimerization; D000076122:Disks Large Homolog 4 Protein; D049029:Dystrophin-Associated Proteins; D016147:Genes, Tumor Suppressor; D005998:Glycine / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008239:Lysine / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D062826:Zonula Occludens-1 Protein
chemical_list: D002352:Carrier Proteins; C114461:DLG5 protein, human; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D049029:Dystrophin-Associated Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D025521:Tumor Suppressor Proteins; D062826:Zonula Occludens-1 Protein; C033652:postsynaptic density proteins; C091824:syntrophin; D001224:Aspartic Acid; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D008239:Lysine; D005998:Glycine