Cryo-EM and NMR Reveal a Membrane-Gated Doc2B-Munc13-1 Complex and a Helical Doc2B-SNARE-Binding Interface

Doc2s modulate neurotransmitter release through interactions with Munc13-1, SNAREs, lipids, and Ca²⁺, which involve the Doc2 C₂ domains and Munc13-interacting domain (MID). However, it is unclear how these interactions are coupled, how Doc2s bind to Munc13-1, and how they bind to the SNAREs. Here we determined the cryo-electron microscopy structure of a Doc2B-Munc13-1 complex aided by an AI-designed Munc13-1 binder, revealing how Doc2B MID binds to a Munc13-1 hydrophobic pocket essential for opening syntaxin-1 and explaining why Doc2B inhibits SNARE assembly. NMR and fluorescence spectroscopy show that Doc2B MID binds lipids and that MID-lipid and MID-Munc13-1 interactions are mutually exclusive, suggesting that membrane binding helps release the inhibition of Munc13-1. NMR shows that Doc2B binds to the SNARE complex via two helices of C₂B. Based on these results and liposome fusion assays, we propose a working model whereby: 1. Doc2B MID-Munc13-1 interactions facilitate synergistic binding of both proteins to the plasma membrane, with the MID inhibiting Munc13-1. 2. Membrane binding facilitates MID-lipid interactions that transiently relieve the inhibition of Munc13-1, allowing SNARE complex assembly. 3. Doc2B binding to the SNARE complex inhibits neurotransmitter release but generates a primed state that mediates neurotransmitter release by an as-yet-unknown mechanism.