RT Journal Article SR Electronic T1 Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.07.05.451112 DO 10.1101/2021.07.05.451112 A1 Manindra Bera A1 Sathish Ramakrishnan A1 Jeff Coleman A1 Shyam S. Krishnakumar A1 James E. Rothman YR 2021 UL http://biorxiv.org/content/early/2021/07/05/2021.07.05.451112.abstract AB Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.Competing Interest StatementThe authors have declared no competing interest.