Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state

Abstract

The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9–15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.

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Figure 1: CPX affects the structural-energetic landscape of SNAREpins as they assemble across membranes.
Figure 2: CPX allows SNAREpins to assemble at a larger distance.
Figure 3: CPX activates and clamps SNAREpin assembly.
Figure 4: Affinity of CPX variants for the SNAREpin.
Figure 5: CPX reshapes the energy landscape of SNAREpin folding.
Figure 6: CPX directly interacts with membrane-anchored t-SNAREs.

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Acknowledgements

This work was supported by the Human Frontier Science Program, Agence Nationale de la Recherche (ANR) Physique et Chimie du Vivant (PCV) grant ANR-08-PCVI-0014 to F.P., US National Institutes of Health grants to J.E.R. and a Partner University Funds exchange grant between the Yale and Ecole Normale Supérieure laboratories. D.T. is funded by the ANR Jeunes Chercheuses et Jeunes Chercheurs (JCJC) grant ANR-09-JCJC-0062-01. We thank T. Melia for many helpful discussions, as well as J. Coleman, W. Eng and A. Garcia-Diaz for technical help.

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F.L. and C.G.G. made constructs and did protein purification. F.L. carried out SFA and ITC measurements. C.G.G. did cell-cell fusion assay. F.L., F.P. and D.T. analyzed the data. F.L., F.P., E.P., D.T. and J.E.R. interpreted the results and prepared the manuscript.

Corresponding author

Correspondence to James E Rothman.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5, Supplementary Table 1, Supplementary Discussion and Supplementary Methods (PDF 1120 kb)

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Li, F., Pincet, F., Perez, E. et al. Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state. Nat Struct Mol Biol 18, 941–946 (2011). https://doi.org/10.1038/nsmb.2102

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