Ligand binding to the PDZ domains of postsynaptic density protein 95

Abstract

Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i)N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain.

Divergent evolution of a protein-protein interaction revealed through ancestral sequence reconstruction and resurrection

“…The binding of H. sapiens CRIPT to H. sapiens DLG4 PDZ3 is well characterized (Niethammer et al 1998;Saro et al 2007;Gianni et al 2011;Toto et al 2016;Doyle et al 1996), and we report a similar binding affinity (0.4 µM) as previous studies ( Fig. 3 and 5, Table 1 and 2).…”

“…This is an interaction that is well studied both from a structural and biophysical point of view, since it was the first structure solved of a PDZ domain with peptide ligand (Doyle 1996). Full affinity of CRIPT (K d in the low µM range for the Homo sapiens CRIPT:DLG4 PDZ3 interaction) is obtained with the six last amino acid residues (Saro et al 2007;Gianni et al 2011;Toto et al 2016).…”

Supertertiary protein structure affects an allosteric network

“…Residues outside the classical PBM influence the binding too, especially P -5 (Tyr) in the case of PDZ3:CRIPT (31,32). However, beyond P -5 the affinity is not significantly affected by the length of CRIPT (33). However, it has not been reported if and how coupling free energies in PDZ and PSG are dependent on peptide ligand length.…”

“…Dansyl-labeled CRIPT peptides were ordered (Zhejiang Ontores Biotechnologies) or synthesized manually by solid-phase peptide synthesis as described previously ( 33 ). Concentrations were measured at 340 nm using an extinction coefficient of 3,400 M −1 ⋅cm −1 ( 50 ).…”

Identifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95

“…The structures of all five subdomains are known 14 , 17 , 18 . Moreover, the PDZ domains of PSD-95 bind critical synaptic proteins such as ionotropic glutamate receptors, neuronal nitric oxide synthase 19 , the synaptic adhesion-protein neuroligin and synGAP, a GTPase linked to synaptic plasticity 12 . Previously, we probed the supertertiary structure of PSD-95 with single-molecule FRET 20 revealing that the first two PDZ domains form a structurally independent supramodule 21 .…”