Dynamic Organization In The Supertertiary Structure Of Pdz3-Sh3-Guk Core Supramodule Of Psd-95 Scaffold Protein

PSD-95, a member of the membrane-associated guanylate kinase (MAGUK) family, is a scaffold protein responsible for the clustering of ionotropic glutamate receptors and ion channels on the postsynaptic membrane of the excitatory synapse. The interdomain organization within the supertertiary structure of PSD-95 reveals two independent supramodules, PDZ1-PDZ2 tandem and PDZ3-SH3-GuK (PSG) core, which are conserved within the synaptic MAGUK homologs. Uncovering the inter-domain interactions and dynamic associations within each supramodule is crucial for elucidating the functional role of PSD-95 in molecular scaffolding and signal transduction. Here, we study the supertertiary structure of the PSG core of PSD-95 by combining replica exchange molecular dynamics simulations with single-molecular FRET (smFRET), Multiparameter Fluorescence Detection (MFD), and disulfide mapping gel assays. Computationally derived inter-residue distances were consistent with smFRET measurements for a large set of 44 inter-dye labeling sites with a Pearson correlation coefficient of ∼0.71. Free energy analysis of DMD simulations revealed three distinct conformational states, where the lowest energy basin had PDZ3 binding to the α-helix of SH3 along its canonical CRIPT binding interface. PDZ3 was also observed to bind GuK in simulations without competing with the MAP1a binding site in the GuK. The third state corresponded to an extended state with PDZ3 dissociated from SH3-GuK. These identified conformational states not only agreed with MFD measurements, but were also confirmed by disulfide mapping experiments where double-cysteine mutants were introduced according to computationally derived supertertiary structures of PSG. Our results underscore the predictive power of the hybrid approach combining DMD simulations with multi-scale FRET experiments and offered structural and dynamic insights to the role of PSG supramodule in PSD-95 scaffolding and signal transduction.