Architecture of the Gβγ-prefusion SNARE complex reveals the molecular mechanism of inhibition of vesicle fusion

ABSTRACT Presynaptic inhibitory GPCR (G i/o GPCR) signaling is an essential regulatory mechanism in vertebrate physiology. Near the presynaptic active zone, G i/o GPCR activation releases G-protein βγ heterodimers (Gβγ) which act to inhibit synaptic vesicle fusion through either modulation of Ca 2+ entry via voltage-gated Ca 2+ channels, or by direct interactions with the core exocytotic machinery comprised of the ternary SNARE complex downstream of Ca 2+ influx. The precise molecular mechanism underlying Gβγ-SNARE mediated inhibition has remained unclear due to lack of structural data for the Gβγ-SNARE complex. We address this long-standing question here by stabilizing the interaction between Gβ1γ2 and a pre-fusion ternary SNARE mimetic and determining the structure using single-particle cryo-EM. We used our cryo-EM envelope to build an atomic level prediction of the interaction interface. We validated key interaction sites predicted by our model at the C-terminus of SNAP-25 using site directed mutagenesis and biochemical affinity measurements. Additionally, we found that Gβ1γ2 and a fragment of the regulatory protein complexin can engage the pre-fusion SNARE complex simultaneously. On the basis of these results, we propose a model in which Gβ1γ2 acts on the partially zipped SNARE complex at a late stage in the vesicle docking and priming cycle. In the model, the amino-terminal coiled-coil of Gβ1γ2 forms an interface with the C-terminus of the target membrane SNARE (t-SNARE) complex to prevent complete incorporation of the vesicle SNARE (v-SNARE) into the core SNARE helical bundle, thus blocking vesicle approach to the plasma membrane. The β-propeller domain of Gβ1 may also sterically hinder vesicle approach. Together these results provide crucial structural insights into the mechanism of binding of Gβγ to the SNARE complex, and lends essential insights into the critical role of GPCR signaling to the SNARE complex in modulating synaptic vesicle fusion.