A Thermodynamic Ligand Binding Study of the Third PDZ Domain (PDZ3) from the Mammalian Neuronal Protein PSD-95

Abstract

The thermodynamic parameters associated with the binding of several series of linear peptides to the third PDZ domain (PDZ3) of the postsynaptic density 95 protein (PSD-95) have been measured using isothermal titration calorimetry (ITC). Two strategies were pursued in developing these binding ligands: (1) systematic N-terminal truncation of sequences derived from the C-terminal regions of identified PDZ3-binding proteins (CRIPT, neuroligin-1, and citron) and (2) selective mutation of specific positions within a consensus hexapeptide (KKETEV) known to bind PDZ3. Each synthetically prepared peptide was used to titrate PDZ3, which yielded the changes in Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) for the binding event. Selected peptides were subjected to additional analysis, which entailed (1) measuring the change in heat capacity (DeltaCp) upon association, to assess the character of the binding interface, and (2) constructing thermodynamic double mutant cycles, to determine the presence of cooperative effects. From the first series, the CRIPT protein proved to be the better source for higher affinity sequences. From the second series, enhanced binding was associated with peptides that closely adhered to the established motif for class I PDZ domain C-termini, X-(T/S)-X-(V/I/L), and more specifically to a narrower motif of X-T-X-V. Further, in both series a length of six residues was necessary and sufficient to capture maximal affinity. In addition, there were significant influences upon binding by modifying the abutting "X" positions. The cumulative results provide greater detail into the specific nature of ligand binding to PDZ3 and will assist in the development of selective molecular probes for the study of this and structurally homologous PDZ domains.

The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

“…The weak binding interaction between JAM-A and PDZ3 was not unanticipated and is well within range of binding affinities observed between PDZ3 of MAGUK proteins and their ligands (32,33). The tight junction is a highly dynamic structure (34,35), and it had been demonstrated that there is a high turnover of ZO-1 and of its binding partners like claudin and occludin in stable epithelial monolayers (35,36).…”

Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding

“…15 Their results showed that residues 0 to -5 (See The additional C-terminal extension is not unique for PDZ3. 25,26 In MAGI PDZ1 for example, an extended C-terminal loop makes direct interactions with Arg -4 , Arg -5 and Thr -6 of the peptide ligand and mutation in this C-terminal loop decreases the binding affinity drastically.…”

“…13,15 But, since the PDZ domain ligand is often very flexible, it has the possibility to wrap around the surface of the PDZ to make additional favorable interactions outside of the canonical binding groove. This binding groove is typically occupied by only four ligand residues, as defined by hydrogen bonds between backbones of the peptide and the second β-strand of the PDZ domain (see Fig.…”

Inhibition of N-Methyl-d-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential

“…To confirm that the partial attenuation of PAR activation by CN2180 is dependent upon the PSD-95/PDZ-binding cyclic peptide KTEV-␤-alanine, we substituted the T and V residues with alanine (KAEA-␤-alanine, CN3200 (Fig. 1), designed to render the myristoylated cyclic peptide ineffective in binding to PSD-95 (30,31). Results show that CN3200 up to 6 nmol had no effect in attenuating NMDA-induced PAR activation (Fig.…”