Interdomain Dynamics Underlie Function and Regulation of Postsynaptic Density Protein 95

Abstract

Postsynaptic Density Protein 95 (PSD95) is a MAGUK-family scaffolding protein found in the excitatory postsynaptic density (PSD). PSD95 plays key roles in regulating synaptic organization and strength through interactions with neuroreceptors at the postsynaptic membrane. Consisting of 5 subdomains (3 PDZ domains, and SH3 domain, and a catalytically-inactive guanylate kinase (GuK) domain) connected by flexible polypeptide linkers, PSD95 further adopts a supertertiary structure in which the first two PDZ domains and the latter three domains partition into two supramodules (PDZ1-2 tandem and PSG supramodule). However, while the structures of the subdomains of PSD95 are well-studied, the underlying mechanisms governing PSD95 function and regulation are not entirely understood. A key characteristic of PSD95 is its structural dynamics. Dynamic intramolecular interactions between the five subdomains provide an attractive explanation for a degree of self-regulation of PSD95 that could extend to other PDZ-containing MAGUKs. In the present work we report a study of full-length PSD95, utilizing an integrative approach combining single-molecule FRET and multiparameter fluorescence detection (MFD) with discrete molecular dynamics (DMD) simulations and using biochemical methods for cross-validation. We analyzed 16 FRET variants to probe dynamics between pairwise combinations of PSD95 subdomains within and between the supramodules. Several key conclusions were drawn from this study: i) we corroborate previous results for the PDZ1-2 tandem identifying fast dynamics between two distinct conformations, now using full-length PSD95, ii) we characterize distinct dynamics within the supramodules and spanning the full PSD95, and iii) we identify three distinct, dynamically interacting structural conformations in the PSG supramodule via DMD and FRET screening. Our findings provide insights into how the interplay between structure and dynamics give rise to biomolecular function and regulation for PSD proteins.