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

ABSTRACT Doc2s modulate neurotransmitter release through interactions with Munc13‐1, SNAREs, lipids, and Ca 2+ , which involve the Doc2 C 2 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 C 2 B. 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.