Structural and Dynamic Features Underlie the Switch of Ligand Binding Specificity in a Tiam1 PDZ Domain Mutant

Abstract

The T-cell lymphoma invasion and metastasis (Tiam) family of proteins are guanine exchange factors (GEFs) for the Rho-family GTPase Rac1 crucial for cell-cell adhesion and cell migration. Deregulation of Tiam1/Rac1 signaling leads to various malignancies, including cardiovascular disease and cancer. Tiam proteins contain several protein-protein interaction domains, in particular a PDZ domain. Previously we found that the Tiam1 and Tiam2 PDZ domains had distinct binding specificities. Intriguingly, four residues in the ligand binding pocket were not conserved between the Tiam1 and Tiam2 PDZ domains. To test their importance in specificity, we engineered a quadruple mutant of the Tiam1 PDZ domain (PDZ-QM), where four residues in the Tiam1 PDZ domain were substituted for those in Tiam2. Remarkably, the Tiam1 PDZ-QM binding preference was changed to that of Tiam2. Here, we used equilibrium binding experiments and structural analyses to investigate the origins for this altered specificity. Ligand-free and -bound PDZ-QM crystal structures showed that enlarged P0 and P-2 ligand binding pockets and a favorable electrostatic interaction at the P-4 sub-pocket were critical for the changed specificity. Biochemical studies indicated that Tiam1 PDZ-QM was less thermally stable than the WT, while NMR studies showed that a set of residues explored multiple conformations. Backbone (15N) and side chain methyl (13C) NMR relaxation studies confirmed the dynamic features of the ligand free Tiam1 PDZ-QM domain. In the presence of ligand, however, PDZ-QM dynamics were dampened. These studies provide novel insights into the structural and dynamic basis for Tiam1 and Tiam2 PDZ domain specificity.