TY - JOUR T1 - The Accessory Helix of Complexin Stabilizes a Partially Unzippered State of the SNARE Complex and Mediates the Complexin Clamping Function <em>In Vivo</em> JF - eneuro JO - eNeuro DO - 10.1523/ENEURO.0526-20.2021 VL - 8 IS - 2 SP - ENEURO.0526-20.2021 AU - Joshua Brady AU - Alexander Vasin AU - Maria Bykhovskaia Y1 - 2021/03/01 UR - http://www.eneuro.org/content/8/2/ENEURO.0526-20.2021.abstract N2 - Spontaneous synaptic transmission is regulated by the protein complexin (Cpx). Cpx binds the SNARE complex, a coil-coiled four-helical bundle that mediates the attachment of a synaptic vesicle (SV) to the presynaptic membrane (PM). Cpx is thought to clamp spontaneous fusion events by stabilizing a partially unraveled state of the SNARE bundle; however, the molecular detail of this mechanism is still debated. We combined electrophysiology, molecular modeling, and site-directed mutagenesis in Drosophila to develop and validate the atomic model of the Cpx-mediated clamped state of the SNARE complex. We took advantage of botulinum neurotoxins (BoNTs) B and G, which cleave the SNARE protein synaptobrevin (Syb) at different sites. Monitoring synaptic depression on BoNT loading revealed that the clamped state of the SNARE complex has two or three unraveled helical turns of Syb. Site-directed mutagenesis showed that the Cpx clamping function is predominantly maintained by its accessory helix (AH), while molecular modeling suggested that the Cpx AH interacts with the unraveled C terminus of Syb and the SV lipid bilayer. The developed molecular model was employed to design new Cpx poor-clamp and super-clamp mutations and to tested the predictions in silico employing molecular dynamics simulations. Subsequently, we generated Drosophila lines harboring these mutations and confirmed the poor-clamp and super-clamp phenotypes in vivo. Altogether, these results validate the atomic model of the Cpx-mediated fusion clamp, wherein the Cpx AH inserts between the SNARE bundle and the SV lipid bilayer, simultaneously binding the unraveled C terminus of Syb and preventing full SNARE assembly. ER -