TY - JOUR
T1 - Subtle interplay between synaptotagmin and complexin binding to the SNARE complex
AU - Xu, Junjie
AU - Brewer, Kyle D.
AU - Perez-Castillejos, Raquel
AU - Rizo-Rey, Jose
N1 - Funding Information:
We thank Yilun Sun for expert technical assistance on protein expression and purification, Kate Lubby-Phelps for help with the use of the TIRF microscope and Thomas Südhof for fruitful discussions. Parts of this research were supported by faculty startup to R.P.-C. from the New Jersey Institute of Technology. This work was supported by grant I-1304 from the Welch Foundation and grant NS40944 from the National Institutes of Health (to J.R.).
PY - 2013/9/23
Y1 - 2013/9/23
N2 - 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 Nand/ 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.
AB - 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 Nand/ 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.
KW - Neurotransmitter release
KW - Protein-membrane interactions
KW - Protein-protein interactions
KW - Synaptic vesicle fusion
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U2 - 10.1016/j.jmb.2013.07.001
DO - 10.1016/j.jmb.2013.07.001
M3 - Article
C2 - 23845424
AN - SCOPUS:84883308384
SN - 0022-2836
VL - 425
SP - 3461
EP - 3475
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 18
ER -