Khan Yousuf A, White K Ian, Pfuetzner Richard A, Singal Bharti, Esquivies Luis, Mckenzie Garvey, Liu Fang, DeLong Katherine, Choi Ucheor B, Montabana Elizabeth, Mclaughlin Theresa, Wickner William T, Brunger Axel T
Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.
Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.
Nat Struct Mol Biol. 2025 Jul 2. doi: 10.1038/s41594-025-01590-w.
SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) proteins drive membrane fusion at different cell compartments as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ (ATPase associated with diverse cellular activities) protein Sec18/NSF and its adaptor Sec17/α-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains. Previous structures of the NSF-α-SNAP-SNARE complex revealed binding to the D1 ATPase pore, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems. Using mass spectrometry in yeast cells, we show N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. We present cryo-electron microscopy (cryo-EM) structures of a yeast SNARE complex, Sec18 and Sec17 in a nonhydrolyzing condition, which show SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side loading and unloading topologically constrained SNARE substrates.
SNARE（可溶性N - 乙基马来酰亚胺敏感因子（NSF）附着蛋白受体）蛋白在不同细胞区室驱动膜融合，因为它们的核心结构域拉链式形成平行的四螺旋束。融合后，这些束被AAA +（与多种细胞活动相关的ATP酶）蛋白Sec18 / NSF及其衔接蛋白Sec17 /α - SNAP拆解，以便它们可用于后续的膜融合轮次。SNARE结构域通常侧翼为C末端跨膜结构域或N末端结构域。NSF - α - SNAP - SNARE复合物的先前结构显示与D1 ATP酶孔结合，这构成了一种拓扑限制，因为SNARE跨膜结构域会像其他AAA +系统所提示的那样阻止底物的完全穿入。利用酵母细胞中的质谱分析，我们展示了N末端SNARE结构域与Sec18的相互作用，加剧了这个拓扑问题。我们展示了在非水解条件下酵母SNARE复合物、Sec18和Sec17的冷冻电子显微镜（cryo - EM）结构，这些结构显示SNARE Sso1穿入Sec18的D1和D2 ATP酶环，其折叠的N末端Habc结构域与D2环相互作用。该结构域在Sec18 / NSF活性过程中不会展开。水解条件下的冷冻电子显微镜结构揭示了Sec18的底物释放和无底物状态，ATP酶环侧面有协同的开放。因此，Sec18 / NSF通过底物侧向加载和卸载拓扑受限的SNARE底物来发挥作用。
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