title: Subtle Interplay between synaptotagmin and complexin binding to the SNARE complex.
abstract: Ca²⁺-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca²⁺ sensor synaptotagmin-1 and on complexins, which play active and inhibitory roles. Release of the complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of complexin-I and a fragment containing the synaptotagmin-1 C2 domains (C2AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C2AB and complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C2AB can displace a complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26-83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26-83, and it is not displaced by C2AB. These results show that interactions of N- and/or C-terminal sequences of complexin-I with the SNARE complex and/or phospholipids increase the affinity of complexin-I for the SNARE complex, hindering dissociation induced by C2AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the complexin-I inhibitory helix without inducing complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and complexin-I in triggering release.
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
authors.forename: Junjie
authors.initials: J
authors.lastname: Xu
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Brewer
authors.forename: Raquel
authors.initials: R
authors.lastname: Perez-Castillejos
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D012995:Solubility; D050857:Synaptotagmins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; C095992:complexin I; D050857:Synaptotagmins; D002118:Calcium
keywords: Ca(2+) triggering; HMQC; HSQC; ITC; MALS; TCEP; TIRF; TROSY; heteronuclear multiple quantum coherence; heteronuclear single quantum coherence; isothermal titration calorimetry; multiangle light scattering; neurotransmitter release; protein–membrane interactions; protein–protein interactions; synaptic vesicle fusion; total internal reflection fluorescence; transverse relaxation optimized spectroscopy; tris(2-carboxyethyl)phosphine