Lipid Disorder and PIP2-Regulated Clustering of Syntaxin-1 JMD–TMD Regions Govern Membrane Fusion Competence

Syntaxin-1 (Stx1), a core neuronal SNARE protein, regulates membrane docking and fusion during neurotransmitter release. Although Stx1 clustering is widely observed at presynaptic active zones and in neuroendocrine cells, how Stx1 isoforms and lipid interactions organize the plasma membrane prior to fusion remains unclear. Here, we performed MARTINI coarse-grained molecular dynamics (cgMD) simulations of 1–10 copies of the juxtamembrane and transmembrane domains (JMD–TMDs) of Stx1A and Stx1B embedded in plasma membrane models to investigate membrane lipid disorder, Stx1 JMD–TMD clustering dynamics, and regulation by Stx1 TMD palmitoylation and phosphatidylinositol 4,5-bisphosphate (PIP2). Stx1 JMD–TMD regions induced local lipid disorder associated with hydrophobic mismatch, with Stx1A producing stronger and more spatially extended perturbations than Stx1B. The magnitude of local lipid disorder remained largely independent of clustering and TMD palmitoylation, whereas palmitoylation spatially restricted and PIP2 depletion broadened the radial extent of membrane perturbation. During clustering, Stx1 JMD–TMDs progressively recruited and redistributed PIP2 into local membrane domains, resulting in ~7-fold local enrichment relative to the bulk membrane. Together, these findings support a model where hydrophobic mismatch primarily governs membrane disorder, whereas multivalent PIP2 recruitment and redistribution stabilize higher-order Stx1 oligomers, thereby promoting membrane environments permissive for SNARE-mediated fusion.