title: Structural and thermodynamic analysis of PDZ-ligand interactions.
abstract: Tiam-family guanine exchange proteins are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These modular proteins contain a variety of signaling domains, including a single postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Here, we show how structural and thermodynamic approaches applied to the Tiam1 PDZ domain can be used to gain unique insights into the affinity and specificity of PDZ-ligand interactions with peptides derived from Syndecan1 and Caspr4 proteins. First, we describe a fluorescence anisotropy-based assay that can be used to determine PDZ-ligand interactions, and describe important considerations in designing binding experiments. Second, we used site-specific mutagenesis in combination with double-mutant cycle analysis to probe the binding energetics and cooperativity of residues in two ligand binding pockets (S(0) and S(-2)) that are involved in Tiam1 PDZ-ligand interactions. Peptide ligand binding results and double-mutant cycle analysis revealed that the S(0) pocket was important for Syndecan1 and Caspr4 peptide interactions and that the S(-2) pocket provided selectivity for the Syndecan1 ligand. Finally, we devised a "peptide evolution" strategy whereby a Model consensus peptide was "evolved" into either the Syndecan1 or Caspr4 peptide by site-directed mutagenesis. These results corroborated the PDZ mutational analysis of the S(0) pocket and identified the P(-4) position in the ligand as critical for Syndecan1 affinity and selectivity. Together, these studies show that a combined structural and thermodynamic approach is powerful for obtaining insights into the origin of Tiam1 PDZ-ligand domain affinity and specificity.
authors.affiliation: Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Tyson R
authors.initials: TR
authors.lastname: Shepherd
authors.forename: Ernesto J
authors.initials: EJ
authors.lastname: Fuentes
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005454:Fluorescence Polarization; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D054731:PDZ Domains; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D013816:Thermodynamics
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; D010455:Peptides; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human