Syntaxin 1A transmembrane domain palmitoylation induces a fusogenic conformation

Neurotransmitter release is triggered by the fusion of synaptic vesicles with the plasma membrane, orchestrated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptobrevin 2 (Syb2), syntaxin 1A (Stx1A), and SNAP25. Recent experimental studies showed that Stx1A palmitoylation of C271/C272 promotes spontaneous neurotransmitter release. However, the mechanistic role of SNARE transmembrane domain (TMD) palmitoylation in membrane fusion remains unclear. To investigate the structural and functional implications of TMD palmitoylation, we employed coarse-grained molecular-dynamics simulations with the MARTINI force field. In simulations of individual SNAREs and of SNAP25/Stx1A (t-SNARE) complexes in a membrane, the palmitoyl chains of Syb2 and Stx1A localize to the membrane midplane, with Stx1A palmitoyl chains bending toward the extracellular leaflet. Nonpalmitoylated Stx1A assumed a conformation where the SNARE domain was lying flat, adhering to the intracellular surface of the membrane. Stx1A dual palmitoylation induced dramatic changes, reducing the tilt of its TMD and stabilizing a more upright conformation of its SNARE domain. This conformation resembles the Stx1A conformation in a t-SNARE complex, providing a potential mechanistic explanation of how Stx1A TMD palmitoylation facilitates early steps in SNARE complex formation and thus promotes spontaneous release. In simulations of the late steps of layers 5-8 SNARE complex zippering in a system of 4 SNARE complexes bridging a 10-nm nanodisc and a planar membrane, fusion pores (FPs) spontaneously opened after a few hundred nanoseconds, preceded by distal-leaflet lipid transfer and followed by FP flickering conductance before FP closure. At this stage, Stx1A TMD palmitoylation delayed lipid transfer and FP formation and decreased FP flicker-open times, whereas the palmitoylation of Syb2 did not affect fusion-pore dynamics. These findings suggest that, after facilitation of priming before FP opening, Stx1A TMD palmitoylation directly affects FP dynamics. These results highlight the essential role of SNARE TMD palmitoylation at multiple stages of neurotransmitter release.