Thermodynamic and Dynamic Basis for the Broadened Ligand Specificity of a Tiam2 PDZ Domain Mutant

Abstract

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules that typically recognize their binding partners through the use of two specificity pockets. Here we examine the consequence of mutating four residues in the Tiam2 PDZ domain specificity pockets to produce a quadruple mutant (QM). Equilibrium binding studies show that the specificity of the Tiam2 QM mutant is similar to that seen in the wild type Tiam1 PDZ domain. Isothermal titration calorimetry experiments show a larger entropic contribution to ligand binding in the QM PDZ domain compared to the WT PDZ domain. Double-mutant cycle analysis uncovered cooperativity between residues in the two specificity pockets with respect to both ligand binding and protein folding. NMR-based HSCQ studies reveal that the wild type Tiam2 PDZ has severe line broadening in several loop regions, while the QM PDZ had additional regions of line broadening. However, peptide ligand binding dampens line broadening for both the Tiam2 WT and QM PDZ domains. Finally, CPMG dispersion experiments indicate that the number of residues experiencing micro to millisecond motions is significantly increased in the QM PDZ domain. We propose a model where enhanced dynamics alters the QM PDZ domain conformational ensemble allowing for broader ligand specificity relative to the WT PDZ domain.