Structural and Thermodynamic Origins of Ligand Specificity in Homologous PDZ Domains from the Tiam-Family of Guanine Exchange Factors

Abstract

PSD-95/DlgA/ZO-1 (PDZ) domains are among the most abundant protein-protein interaction domains in the human proteome and typically bind the 4-10 most C-terminal residues of its interaction partner with exquisite specificity. To investigate the origin of this specificity, we used two homologous PDZ domains from the Tiam-family of GEFs that have distinct but overlapping specificity for ligands. The Tiam1 PDZ domain binds 8-residue long C-terminal peptides derived from the proteins Syndecan1 and Caspr4 with micromolar affinity but does not bind Neurexin1. In contrast, the Tiam2 PDZ domain binds to peptides derived from Caspr4 and Neurexin1 with low micromolar affinity but not Syndecan1. Analysis of the X-ray crystal structure of the Tiam1 PDZ domain bound to a “model” peptide shows two specificity pockets created by four residues in the Tiam1 PDZ domain. Moreover, comparison of nuclear magnetic resonance (NMR) titrations of the Tiam1 PDZ domain with the Syndecan1 and Caspr4 peptides showed substantial differences in the changes in chemical shift in these residues. Sequence comparison of Tiam-family PDZ domains revealed that these residues are not conserved, further suggesting that they play a role in establishing ligand specificity. Double mutant cycle analysis of residues in these two pockets revealed ligand-dependent cooperativity, supporting their role in specificity is ligand specific. Remarkably, substitution of all four residues in the Tiam1 PDZ domain with the amino acids found in the Tiam2 PDZ domain switched the specificity to that of Tiam2. Collectively, our data suggest that Tiam-family proteins have highly evolved PDZ-ligand interfaces with distinct specificities, and that they have disparate PDZ-dependent biological functions.