Two Pleckstrin homology domains scaffold SNAP-47-containing SNARE complexes
Long-term potentiation requires the regulated insertion of AMPA receptors into the postsynaptic membrane of neurons, a process mediated by a SNARE complex containing SNAP-47, syntaxin-3, and synaptobrevin-2. Compared to other SNAP proteins, SNAP-47 exhibits slower fusion kinetics and contains uncharacterized regions whose functions remain unclear. Here, we integrated biochemical approaches with artificial intelligence structure prediction tools and molecular dynamics to investigate the architecture of SNAP-47-containing SNARE complexes. AlphaFold3 predictions of the ternary SNAP-47-containing SNARE complex revealed one known Pleckstrin homology domain (PH1) and one previously unidentified PH domain (PH2), which we validated experimentally. BioEmu and molecular dynamics simulations indicated that PH1 and PH2 form a stable and energetically favorable interface. In this bound state, PH1 stably interacted with the SNARE coiled coil, and in silico acetylation at a lysine whose acetylation is known to impair SNAP-47 function disrupted this PH1-SNARE interface. AMPAR insertion via SNAP-47-containing SNARE complexes is triggered by the calcium sensor synaptotagmin, and we observed that PH1, PH2, and synaptotagmin-1 can simultaneously engage with the plasma membrane, consistent with a pro-fusion conformation. Together, we provide new mechanistic insight into how PH domains regulate SNAP-47-mediated membrane fusion during synaptic plasticity.