Structural and thermodynamic origins of distinct ligand specificity of two homologous PDZ domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a previously uncharacterized post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Here we report on the structure, specificity, and function of the Tiam1 and Tiam2 PDZ domains.

A consensus PDZ-binding motif for Tiam1 was used to predict that two cell adhesion proteins, Syndecan 1 (Sdc1) and Caspr4, are potential Tiam1 PDZ domain binding proteins. Binding interactions were confirmed using fluorescence- and NMR- based binding experiments. The Tiam1 PDZ domain in complex with the C-terminal tails of Sdc1 and phosphorylated Sdc1 were solved using X-ray crystallography. Results showed four residues in two binding pockets in the PDZ domain are important for specificity. Cell biological analysis confirmed the Tiam1/Sdc1 interaction and showed that the PDZ domain has a function in cell-matrix adhesion and cell migration.

The four residues deemed important determinants of Tiam1 PDZ domain specificity are not conserved in Tiam2. A combinatorial peptide screen, in combination with biophysical studies, identified a consensus binding sequence for both PDZ domains. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities - the Tiam2 PDZ domain was found to bind Caspr4 and neurexin1 but not Sdc1. Additionally, the Tiam2 PDZ domain exhibits significant flexibility in two different regions, a feature not seen in Tiam1.

Double-mutant cycle analysis of the four important residues revealed ligand- dependent energetic couplings. Mutating all four residues switched the ligand specificity to that of Tiam2. Analysis of Tiam-family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. A set of "evolved peptides" was used to show the PDZ domain interactions are cooperative throughout the binding pocket in a ligand-specific manner. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain/ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.