Palmitoylation of PSD95 flips the bilayer juxtaposed domain and drives cluster formation on membrane

Postsynaptic density protein 95 (PSD95) is a ubiquitous and highly abundant scaffolding protein in excitatory neurons. It plays a critical role in organizing the molecular structure and function of synapses by clustering at the postsynaptic density (PSD), where it anchors neurotransmitter receptors, ion channels, and signaling enzymes. These clusters form a dynamic nanodomain molecular platform that is essential for synaptic transmission and plasticity. Disruption of PSD95 clustering affects synaptic organization and is involved in various neuropsychiatric disorders. Palmitoylation, a reversible post-translational modification involving the addition of a palmitate group to specific Cysteine residues, has been experimentally linked to PSD95 membrane localization and clustering. However, the structural and mechanistic basis of this process remains poorly understood. Using a multiscale molecular dynamics approach that combines all-atom and coarse-grained simulations, we investigated how palmitoylation influences the conformational dynamics, membrane association, and self-assembly of PSD95. Our simulations reveal that palmitoylation induces PSD95 to adopt and maintain an open conformation in solution as well as stabilizes its assembly-primed extended configuration when bound to the membrane. Specifically, in the unmodified state, the N-terminal PDZ1 domain remains distal to the membrane, while PDZ2 preferentially engages with it. Palmitoylation flips this arrangement and anchors PDZ1 to the membrane, thereby promoting an extended configuration conducive to protein-protein interactions and clustering. We also find that membrane lipid composition modulates this behavior. Simulations with complex synaptic membranes compared to POPC-only membranes suggest that negatively charged lipids influence domain orientation through electrostatic interactions, working in combination with palmitoylation to shape PSD95 conformational space. Finally, coarse-grained simulations show that palmitoylated PSD95 molecules can stably associate on the membrane surface, whereas unmodified molecules fail to form persistent dimers. Together, our results provide a mechanistic model for how palmitoylation facilitates early-stage clustering of PSD95, offering insights that are difficult to capture through experimental approaches alone.