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

Postsynaptic density protein 95 (PSD95) is a highly abundant scaffolding protein at excitatory synapses, where it organizes the molecular architecture of the postsynaptic density (PSD) by anchoring neurotransmitter receptors, ion channels, adhesion molecules, and signaling enzymes. Emerging evidence indicates that PSD95 forms transient “nanodomains” essential for the fidelity of synaptic transmission and plasticity, and disruptions in its clustering are linked to neuropsychiatric disorders. Palmitoylation, a reversible post-translational modification involving the covalent attachment of palmitate to Cysteine residues, has been implicated in the localization and clustering of the PSD95 at the plasma membrane, but its structural and mechanistic role remains unclear. Here, we use a multiscale molecular dynamics approach that combines all-atom and coarse-grained simulations to investigate how palmitoylation regulates PSD95 conformational dynamics, membrane association, and self-assembly. Our results show that palmitoylation promotes an open conformation in solution and stabilizes an extended assembly-primed state at the membrane. In the unmodified state, the PDZ1 domain remains distal while PDZ2 preferentially engages with the membrane; palmitoylation reverses this arrangement, anchoring PDZ1 to the membrane and enhancing clustering propensity. Membrane lipid composition further modulates this behavior, with negatively charged lipids influencing favorable domains orientation through electrostatic interactions. Finally, coarse-grained simulations reveal that palmitoylated PSD95 forms stable dimers on the membrane surface, whereas unmodified molecules fail to associate persistently. Together, these findings provide a mechanistic model for how palmitoylation facilitates the early stages of PSD95 clustering, offering nanoscale insights into synaptic organization that complement experimental observations.