FC0095
Postsynaptic density 95 (PSD-95) PDZ3  -  PSD-95 SH3-GK

Biological function
PSD-95 is highly abundant in the postsynaptic density of excitatory neurons and is responsible for coupling glutamate receptors with internal postsynaptic structures. It is one of the best known members of the membrane-associated guanylate kinase (MAGUK) scaffold proteins that organize events of signal transduction, cell adhesion, and molecular trafficking at specialized cell-cell junctions such as synapses and tight junctions.

Domain organization/sequence features
Multiple protein-protein interacting domains connected by variable length linkers, usually three N-terminal PDZ domains followed by an SH3-guanylate kinase (GK) domain module.

Structural evidence
SAXS suggests that PDZ3 contacts SH3-GK, but PDZ3 is not rigidly locked against SH3-GK. chemical shift perturbations (CSPs) of PDZ3 due to the presence of SH3 were modest, consistent with a weak interdomain interaction. iPRE measurements and EOM-SAXS analysis consistently show a dynamic interaction between PDZ3 and SH3-GK. Addition of CRIPT peptide to PDZ3-SH3 reduced the iPREs at residues proximal to the PDZ3 peptide binding groove indicating movement of this binding site away from the SH3 spin label. CRIPT binding also serves to rotate PDZ3 to bring other residues slightly closer, on average, to SH3. Ala mutation of L411 and M412 at the central part of the linker increases ¹⁵N T₂ relaxation rates by 10-15 ms by incresed tumbling of PDZ3. This is consistent with the model that binding of the linker hydrophobic motif mediates the dynamic association between PDZ3 and SH3-GK.

Biochemical evidence
Gly-Ser substitutions in residues 396–401 and 411–424 in the linker of our PDZ3-SH3 construct has a near- identical effect to CRIPT binding, reducing iPREs at the binding face and reorienting PDZ3 relative to SH3. Thus CRIPT binding and linker replacement disrupt the weak, but specific, interaction of PDZ3 and SH3-GK.

Structure/Mechanism
PSD-95 MAGUK core is intrinsically unstable. While there is a heterogeneous ensemble of interdomain positions, the dominant configuration is for PDZ3 contacting the SH3-GK module via its C-terminal ligand binding groove. Hydrophobic side chains of L411-M412, which sit in the central portion of the linker, insert into the PDZ ligand binding pocket. An overall compact arrangement of PSG domains in PSD-95 and indicate that the active site of PDZ3 binds protein ligands for both intra- and interdomain assembly. The involvement of the PDZ3 active site in the interdomain interaction shows how binding of CRIPT peptide displaces SH3-GK resulting in the more expanded form. Linker mediated interactions serve to shorten the overall length of the C-terminal half of PSD-95: the PDZ domain of the MAGUK core approaches the SH3-GK module, stabilizes domain-domain interactions and sequesters the Hook subdomain. This can result an overall shortening of the PSD-95 molecule, by as much as 50Å.

Mechanism category
tethering

Posttranslational modification
S415 and S418, which have been shown to be phosphorylated are in proximity to three consecutive, negatively charged SH3 residues (D467, E468, and E469), suggesting a possible mechanism for phosphorylation-induced disruption of the PDZ3-SH3 interaction.

Significance
Fuzziness mediates allosteric communication between the PDZ3 and GK domains and serve to regulate the overall length of PSD-95, which may be an important parameter for dynamic remodeling of the PSD.

Medical relevance
Decreased levels of functional PSD-95 are associated with autism spectrum disorders (ASDs) and disorders of the central nervous system such as Alzheimer’s disease and neuropathic pain, and most recently PSD-95 has been identified as a promising target for the treatment of stroke.