Complexin cross-links prefusion SNAREs into a zigzag array


Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

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Figure 1: Structure of the prefusion CPX–SNARE complex.
Figure 2: Interacting surfaces of CPXacc and the t-SNAREs.
Figure 3: Characterization of the interaction of CPXacc with SNARE complexes by isothermal titration calorimetry.
Figure 4: FRET experiments probing CPX orientation in pre- and postfusion CPX–SNARE complexes.
Figure 5: Effects of CPX and VAMP2 mutations on clamping in cell-cell fusion assays.
Figure 6: Molecular models for CPX clamping.

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We wish to thank the staffs of beamline X29 at the National Synchrotron Light Source, Brookhaven National Laboratory and of the Northeastern Collaborative Access Team (NE-CAT) facility at the Advanced Photon Source, Argonne National Laboratory, for their help in data collection; L. Khandan (Yale University) and S. Baguley (Yale University) for technical assistance; and J. Coleman (Yale University) for advice. We are grateful to E. Karatekin (Yale University) and D.W. Rodgers (University of Kentucky) for discussions regarding this manuscript. This work was supported by grants from the US National Institutes of Health to K.M.R. (R01GM080616) and to J.E.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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D.K. coordinated the experiments in this paper, was responsible for structure analysis and designed constructs for the functional analyses. S.S.K. and D.T.R. conducted the FRET experiments; F.L. conducted the ITC analysis and C.G.G. carried out the cell-cell fusion experiments. F.P. contributed to the analysis of the FRET and ITC data. D.K., J.E.R. and K.M.R. analyzed data and wrote this manuscript.

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Correspondence to James E Rothman or Karin M Reinisch.

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Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Methods (PDF 1031 kb)

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Kümmel, D., Krishnakumar, S., Radoff, D. et al. Complexin cross-links prefusion SNAREs into a zigzag array. Nat Struct Mol Biol 18, 927–933 (2011).

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