Entry created on 1 July 2019 (Revision 1.0) Annotator: Bálint Mészáros
This entry is part of a multi-component system encompassing the following entries: J3QQ18 P78352-3
Basic protein information
Accession P78352-3
Common name PSD-95
Gene DLG4_HUMAN
Organism Homo sapiens
Uniprot name Isoform 3 of Disks large homolog 4
Basic LLPS information
Organelle postsynaptic density
Type of experimental evidence
Joined entry J3QQ18 P78352-3
Protein region(s) mediating LLPS
306
-
721
C-terminal "PSG" region (composd of a PDZ domain, an SH3 domain, and a guanylatekinase domain)
Based on the experimental results of the following publication: 27565345
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
SynGAP forms a stable trimer in solution. Trimeric SynGAP CC-PBM (coiled-coil domain and the following PDZ binding motif) recruits two PSD-95 PSG (3 PDZ+SH3+GK domains) to form a 3:2 complex. The SynGAP/PSD-95 3:2 complex undergoes LLPS and evolves into a postsynaptic density (PSD). PSDs are assemblies responsible for receiving, interpreting and storage of signals transmitted by presynaptic axonal termini. They are disc-shaped, electron-dense thickenings that contact with postsynaptic membranes on its one face and with cytoplasm on the other face, forming semi-open mesoscale cellular compartments. Mutations altering the SynGAP/PSD-95 interaction can contribute to various brain disorders, including autism and IDs (intellectual disordes) (PMID:27565345).
Literature supporting the LLPS: 28524815, 27565345
Functional class of membraneless organelle: activation/nucleation/signal amplification/bioreactor; sensor
Binding partners (at biological protein concentrations)
1) SynGAP coiled-coil/PDZ binding motif fragment (strictly required)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required.
Molecular interaction types contributing to LLPS
multivalent domain-motif (PMID:27565345)
Determinants of phase separation and droplet properties
1) protein concentration 2) temperature
Membrane cluster No
Partner-dependent Yes
RNA-dependent No
PTM required No
Domain-motif interactions Yes
Discrete oligomerization Yes
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
Physical interaction between SynGAP and PSD-95 was verified in vitro using purified proteins. Isothermal titration calorimetry (ITC)-based titration assay showed that N-terminal thioredoxin-tagged last 30 residues of SynGAP binds to each of the three PDZ domains of PSD-95 with comparable and weak affinities, and this interaction is enhanced by the alpha helix following the third PDZ domain. Similar in vitro analysis using fast protein liquid chromatog-raphy (FPLC) coupled with a static light-scattering assay and circular dichroism revealed that SynGAP forms a paraller coiled-coil trimer. In vitro structural studies also showed that trimeric SynGAP CC-PBM (residues 1147-1308) recruits two PSD-95 PSG (306-721) to form a 3:2 stochiometric complex (physical interaction). In vitro mixing of purified SynGAP CC-PBM andPSD-95 PSG above certain concentrations caused the sample solutions to become opalescent immediately owing to the formation of small spherical (morphology) droplets spanning various diameters (particle size and count), as followed using light microscopy. Neither SynGAP CC-PBM nor PSD-95PSG alone could form condensed liquid phase. Liquid-to-liquid phase separation and condensed liquid phase droplets fusion of the SynGAP/PSD-95 complex (particle size and count) was followed using Alexa488-labeled SynGAP CC-PBM and Cy3-labeled PSD-95 PSG (fluorescent tagging) by fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) analysis of Cy3-labled PSD-95 droplets demonstrated that PSD-95 molecules constantly exchange between droplets and the surrounding aqueous solution (PMID:27565345). When GFP-SynGAP CC-PBM and RFP-PSD-95 PSG were co-expressed in HeLa cells in vivo, many bright puncta were observed containing both GFP and RFP signals. Both GFP and RFP signal intensities were much brighter within the puncta than the surrounding cytoplasm, indicating enrichment with SynGAP and PSD-95. No puncta were observed in cells when only PSD-95 PSG or SynGAP CC-PBM was expressed (PMID:27565345).
Experimental observations supporting the liquid material state of the condensate
dynamic movement/reorganization of molecules within the droplet (PMID:27565345) dynamic exchange of molecules with surrounding solvent (PMID:27565345) morphological traits (PMID:27565345) temperature-dependence (PMID:27565345)