Eitel Anna R, Mueller Benjamin K, Kaya Ali I, Young Montana, Cassada Jackson B, Bell Eric W, Schnitkey Lauren, Zurawski Zack, Yim Yun Y, Zhou Qiangjun, Meiler Jens, Hamm Heidi E
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA.
Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA.
J Biol Chem. 2025 Jun 14;301(8):110377. doi: 10.1016/j.jbc.2025.110377.
Neurotransmitter release is a complex process involving tightly controlled co-factors and protein-protein interactions. G-protein coupled receptors negatively regulate exocytosis via the interaction of G-protein βγ (Gβγ) heterodimers with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The neuronal ternary SNARE complex comprises synaptosomal-associated protein-25 (SNAP25), syntaxin-1A, and synaptobrevin-2. The regions of the SNARE complex that are important for interactions with Gβγ have been extensively characterized, but the critical sites on Gβγ are not well understood. Furthermore, the molecular basis for the specificity of different Gβ and Gγ isoforms for SNARE proteins remains elusive. Thus, we holistically probed the entire family of human Gβ and Gγ isoforms for regions critical for the target-SNARE (tSNARE) interaction using a peptide screening approach. Gβ and γ peptides with high affinities for tSNARE were then subjected to alanine scanning mutagenesis to identify the interaction sites. We found that the N-terminal coiled-coil domain of Gβγ as well as the β-propeller domain of Gβ are hotspots for SNARE interactions. Additionally, we found that the N-terminal Gγ2 peptide is a potent inhibitor of interactions between full-length Gβ1γ2 and SNAP25. We discovered that Gβ1γ2 preferentially interacts with ternary SNARE in the pre-fusion, partially zipped conformation, likely due to increased exposure of the C-terminus of SNAP25. Our combined results suggest that specific Gβγ heterodimers bind to ternary SNARE in the docked and primed state via critical residues of the β-propeller and N-terminal coil-coil domains. We propose that Gβγ binding disrupts zippering up of the SNARE complex and thereby vesicle fusion.
神经递质释放是一个复杂的过程，涉及严格控制的辅助因子和蛋白质 - 蛋白质相互作用。G蛋白偶联受体通过G蛋白βγ（Gβγ）异二聚体与可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合体的相互作用来负调控胞吐作用。神经元三元SNARE复合体由突触体相关蛋白25（SNAP25）、 syntaxin - 1A和突触小泡蛋白2组成。SNARE复合体中对与Gβγ相互作用重要的区域已得到广泛表征，但Gβγ上的关键位点尚不清楚。此外，不同Gβ和Gγ亚型对SNARE蛋白特异性的分子基础仍然难以捉摸。因此，我们使用肽筛选方法全面探究了人类Gβ和Gγ亚型家族中对靶标SNARE（tSNARE）相互作用至关重要的区域。然后对与tSNARE具有高亲和力的Gβ和γ肽进行丙氨酸扫描诱变，以确定相互作用位点。我们发现Gβγ的N端卷曲螺旋结构域以及Gβ的β - 螺旋桨结构域是SNARE相互作用的热点。此外，我们发现N端Gγ2肽是全长Gβ1γ2与SNAP25之间相互作用的有效抑制剂。我们发现Gβ1γ2优先与预融合、部分拉链化构象的三元SNARE相互作用，这可能是由于SNAP25 C端暴露增加所致。我们的综合结果表明，特定的Gβγ异二聚体通过β - 螺旋桨和N端卷曲螺旋结构域的关键残基与对接和引发状态的三元SNARE结合。我们提出Gβγ结合会破坏SNARE复合体的拉链化，从而阻止囊泡融合。
