Structural and thermodynamic analysis of PDZ-ligand interactions.

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.