A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion

Abstract

The crystal structure of complexin bound to a prefusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an 'open' conformation, binding another SNAREpin and inhibiting its assembly, to clamp fusion. In contrast, the accessory helix in the postfusion complex parallels the SNARE complex in a 'closed' conformation. Here we use targeted mutations, FRET spectroscopy and a functional assay that reconstitutes Ca2+-triggered exocytosis to show that the conformational switch from open to closed in complexin is needed for synaptotagmin-Ca2+ to trigger fusion. Triggering fusion requires the zippering of three crucial aspartate residues in the switch region (residues 64–68) of v-SNARE. Conformational switching in complexin is integral to clamp release and is probably triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore.

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Figure 1: Zippering of one turn of VAMP2 helix triggers the switch in CPX position.
Figure 2: Aspartate residues 64, 65 and 68 on VAMP2 mediate the switch in CPX position.
Figure 3: Interaction of CPXcen with aspartate residues 64, 65 and 68 on VAMP2 provides thermodynamic driving force for the switch.
Figure 4: The switch in CPXacc position is necessary for synaptotagmin-Ca2+ to trigger fusion.
Figure 5: Perturbation of a single SNARE complex in the zigzag array should be sufficient to rapidly disassemble the clamp in response to neuronal stimulus.

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Acknowledgements

We wish to thank T. Melia (Yale University) for critical reading of the manuscript. This work was supported by US National Institutes of Health grants to J.E.R. and K.M.R., an Agence Nationale de la Recherche (ANR) Physique et Chimie du Vivant (PCV) grant to F.P., and a grant from the Deutsche Forschungsgemeinschaft to D.K.

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S.S.K., D.T.R. and D.K. did the mutagenesis and protein purification, S.S.K. and D.T.R. carried out the fluorescence measurements, F.L. took the ITC measurements and C.G.G. did the cell-cell fusion assay. L.K., S.B. and J.C. provided technical assistance. Results were analyzed and discussed by all authors. The manuscript was prepared by S.S.K., D.T.R., K.M.R., F.P. and J.E.R.

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Correspondence to James E Rothman.

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Krishnakumar, S., Radoff, D., Kümmel, D. et al. A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion. Nat Struct Mol Biol 18, 934–940 (2011). https://doi.org/10.1038/nsmb.2103

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