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The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes

Nature Structural & Molecular Biology volume 29, pages 97–107 (2022)Cite this article

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Abstract

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

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Fig. 1: Cpx forms pores in bilayers via its C-terminal amphipathic helix.
Fig. 2: MD simulations of Cpx Ct21 peptides with lipid bilayers.
Fig. 3: Cpx forms pores and remodels GUV membranes.
Fig. 4: Cpx vesiculates, fragments and twists membranes.
Fig. 5: The Cpx C-terminal amphipathic helix stabilizes the open state of nascent fusion pores formed by trans-SNARE complexes.
Fig. 6: Deletion of the C-terminal amphipathic helix converts Cpx to an inhibitor of stable fusion pores.

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Data availability

All graphed raw data are provided as source data. With the exception of RCSB Protein Data Bank to access PDB 1KIL, no specific databases or third party data were used in this study. Source data are provided with this paper.

Code availability

No unique code was used in this study.

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Acknowledgements

We thank the members of the Chapman laboratory and M.B. Jackson for helpful discussions. We also thank A. Ferguson (University of Chicago) for providing access to the AMP prediction tool and S. Mishra for preliminary work performing HEK cell patch-clamp recordings. This work was supported by Pew Charitable Trust grant no. 864K625 (E.R.C. and D.H.), National Institutes of Health grant nos. MH061876 and NS097362 (E.R.C.), P01-GM121203 (N.V.) and DP2GM140920 (H.B.). Equipment for the cryo-CLEM and in situ cellular tomography workflow used in this work was funded by National Institutes of Health grant nos. S10-OD012372 (D.H.), S10-OD026926 (D.H.), P01-GM121203 (N.V.) and R01-AI132378 (N.V., D.H.), and Pew Charitable Trust grant no. 864K625. The computational component is supported by the grant no. NSF-DMS1661900 (Q.C.) Computational resources from the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by NSF grant no. OCI-1053575 (Q.C.), are greatly appreciated; computations are also supported in part by the Shared Computing Cluster, which is administered by Boston University’s Research Computing Services. E.R.C. is an Investigator of the Howard Hughes Medical Institute.

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Author notes

  1. These authors contributed equally: Kevin C. Courtney, Lanxi Wu.

Authors and Affiliations

  1. Howard Hughes Medical Institute and the Department of Neuroscience, University of Wisconsin, Madison, WI, USA

    Kevin C. Courtney, Lanxi Wu, Zhao Zhang, Zhenyong Wu, Mazdak M. Bradberry, Huan Bao & Edwin R. Chapman

  2. Department of Chemistry, Boston University, Boston, MA, USA

    Taraknath Mandal, Mohammad Alaghemandi & Qiang Cui

  3. Department of Physics, Indian Institute of Technology Kanpur, Kanpur, India

    Taraknath Mandal

  4. Scintillon Institute, San Diego, CA, USA

    Mark Swift, Niels Volkmann & Dorit Hanein

  5. Medical Scientist Training Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA

    Mazdak M. Bradberry

  6. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Claire Deo & Luke D. Lavis

  7. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Claire Deo

  8. Structural Image Analysis Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université de Paris, CNRS UMR3528, Paris, France

    Niels Volkmann

  9. Structural Studies of Macromolecular Machines in Cellulo Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université de Paris, CNRS UMR3528, Paris, France

    Dorit Hanein

  10. Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA

    Huan Bao

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Contributions

K.C.C. was involved with the conceptualization; methodology; validation; formal analysis; investigation; original draft preparation, review and editing of the paper and visualization. L.W. was involved with the methodology, formal analysis, investigation, review and editing of the paper and visualization. T.M. was involved with the methodology, formal analysis, investigation, review and editing of the paper and visualization. M.S. was involved with the methodology and investigation. Z.Z. was involved with the formal analysis, investigation, review and editing of the paper and visualization. M.A. was involved with the formal analysis and investigation. Z.W. was involved with the formal analysis and investigation. M.M.B. was involved with the investigation and review and editing of the paper. C.D. was involved with the methodology and resources. L.D.L. was involved with the resources, supervision and funding acquisition. N.V. was involved with the formal analysis, investigation, supervision, review and editing of the paper, visualization and funding acquisition. D.H. was involved with the methodology, formal analysis, investigation, supervision, review and editing of the paper, visualization and funding acquisition. Q.C. was involved with the methodology, supervision and funding acquisition. H.B. was involved with the conceptualization; methodology; validation; formal analysis; investigation; original draft preparation, review and editing of the paper and visualization. E.R.C. was involved with the conceptualization; validation; resources; original draft preparation, review and editing of the paper; supervision and funding acquisition.

Corresponding author

Correspondence to Edwin R. Chapman.

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Extended data

Extended Data Fig. 1 The C-terminal alpha helix of Cpx creates pores in unilamellar vesicles and lyses spheroplasts.

(a) Leakage of glutamate from LUVs composed of PC (black), PC/PS (blue) or total brain lipids (red) after treatment with increasing concentrations of Cpx, Cpx ΔCt21 and Ct21. Glutamate release was monitored via the glutamate sensor, iGluSnFR, in the media (1 µM). (b) Lysis of E. coli spheroplasts with increasing concentrations of Cpx, Cpx ΔCt21 and Ct21 peptide monitored via absorbance at 500 nm. Panels A and B represent the average of three independent experiments. Error bars represent standard error of the mean. (c) Image of the JF635i HaloTag ligand structure. (d) Representative fluorescence emission traces of 5 µM of the JF635i ligand, excited at 635 nm, with and without 5 µM of recombinant HaloTag protein. (e) GUVs labeled with rhodamine-PE (white), and with encapsulated HaloTag protein, before and after treatment with various concentrations of Cpx. JF635i (magenta) is present in the media; this dye strongly fluoresces upon entering GUVs and binding the HaloTag protein. (f) GUV leak assay, described in Fig. 3, performed with 1 µM of multiple Cpx isoforms. (g) GUV leak assay performed with 2 µM of the cytoplasmic domain of synaptotagmin 1 (syt1), bovine serum albumin (BSA), Munc18, α-SNAP, NSF or α-synuclein. Each of the GUV conditions were repeated at least three times.

Source data

Extended Data Fig. 2 Negative-stain TEM images of miscellaneous structures of Cpx-treated LUVs.

(a) A zoom-out image with four distinct features highlighted by colored boxes that were consistently observed from three independent experiments. Each feature belongs to a category of relevant structures, and more representative cropped images are given in panels b-e: (b) mouth-like opening (pink); (c) ring-like attachment (green); (d) discoidal structures (blue); (e) potential vesicle budding intermediates (yellow). Scale bars: 50 nm.

Extended Data Fig. 3 Electron cryo-tomography of liposomes before and after Cpx treatment.

Electron cryo-tomography of 100 nm LUVs with or without treatment of 10 µM Cpx. Three-dimensional surface representations and the corresponding two-dimensional cross sections are shown. In all cases, the scale bar represents 50 nm. The experiment was repeated twice. (a) Representative electron cryo-tomography of untreated 100 nm LUVs. (b) Representative electron cryo-tomography of 100 nm LUVs showing Cpx treatment often induced vesiculation. (c) Representative electron cryo-tomography of 100 nm LUVs showing Cpx treatment commonly causes LUVs to fragment into membranous networks. (d) Representative electron cryo-tomography of 100 nm LUVs showing Cpx treatment can cause LUVs to twist into highly curved structures.

Extended Data Fig. 4 Recombinant Cpx forms pores in the plasma membrane of mammalian cells.

(a) Illustration of the HEK-293T cell patch-clamp setup. A typical cell-attached patch was formed on the surface of the cell, but using a pipette that contained WT full-length or ΔCt21 Cpx. (b) Representative electrophysiological recordings; in 7 out of 21 trials, WT Cpx generated positive currents. In the control or Cpx ΔCt21 conditions, no pores were observed in 5 and 8 trials, respectively. (c) Zoomed-in regions of interest (ROI), from the WT Cpx condition in panel b, showing transient positive currents that precede the large upswing in positive current.

Extended Data Fig. 5 Cpx activity can be regulated by phosphorylation and calmodulin.

(a) Illustration of the GUV leakage assay using Alexa 647 dye. Rhodamine-PE labelled GUVs, pseudo-colored in white, were formed in the presence of 10 µM Alexa 647 dye, followed by iso-osmolar buffer exchange. Upon pore formation, the dye, pseudo-colored in yellow, escapes from the lumen of the GUVs. b) Representative images of Control GUVs and after treatment with 5 µM Cpx (orange), Cpx ΔCt21 (purple), Cpx Ct21 scrambled (beige), Cpx (S115, T119D) (green), 10 µM Calmodulin (CaM) (light gray) or CaM + Cpx in EGTA (blue) and Ca2+ (red). The indicated conditions were independently repeated >3 times with consistent results. The sequence for the Cpx Ct21 scramble is found in Table 1. The white scale bar represents 10 µm. (c) Quantification of GUV lumenal fluorescence after the treatments described in panel B. The data are pooled from three independent experiments and the errors bars represent standard deviation. **** denotes p < 0.0001 and ns denotes not significant, determined by ANOVA analysis and Tukey multiple comparisons test. (d) Illustration of the Cpx-CaM binding assay. A single cysteine variant (L124C) of Cpx was labeled with an environment-sensitive dye, NBD, and the fluorescence emission was monitored by fluorometer. In the presence of Ca2+, CaM binds the Cpx C-terminal helix, causing an increase in fluorescence (e) Representative fluorescence spectrum of Cpx (L124C-NBD). NBD fluorescence in 25 mM HEPES (pH 7.4), 100 mM KCl was monitored without CaM (black) and with CaM in EGTA (blue) or Ca2+ (red). The change in NBD fluorescence in the Ca2+ condition indicates CaM binds the Cpx amphipathic helix.

Source data

Extended Data Fig. 6 Five copies of TMD-Cpx and three copies of TMD-Cpx ∆Ct21-melittin do not form SNARE-independent pores in the ND-BLM system.

(a) Illustration for TMD-Cpx (left panel) and TMD- Cpx ∆Ct21-melittin (right panel). The yellow asterisk indicates residue E114, the beginning of Ct21. (b) Sample trace from TMD-Cpx control experiments in the absence of syb2. Five copies of TMD-Cpx alone (no syb2) were reconstituted in the NDs. NDs with TMD-Cpx alone were added to the BLM system along with t-SNARE-liposomes, no pores formed, after 2 hrs. After NDs with syb2 were introduced, multiple pores opened. (c) Sample trace from TMD-Cpx ∆Ct21-melittin experiments in the absence of t-SNAREs. Three copies of syb2 and three copies of TMD-Cpx ∆Ct21-melittin were co-reconstituted in the NDs. NDs were introduced to the BLM system, no pore formed, after 2 hrs. After t-SNARE liposomes were added to the same system, multiple pores opened.

Extended Data Fig. 7 Truncation of the Cpx C-terminus causes fusion pore instability and reduces SUV lipid mixing.

(a) Crystal structure from pdb 1KIL showing Cpx (shown in red) bound to the SNARE complex formed by syb2 (shown in yellow), SNAP25B (shown in orange) and syntaxin 1a (Syx1a) (shown in green). Cpx residues R48 and R59 are shown in cyan. (b) Lipid mixing assay performed with v-SNARE and t-SNARE containing vesicles over time. The t-SNARE SUVs remain constant, while the v-SNARE SUVs were reconstituted as syb2 alone (grey), syb2 + TMD-Cpx (blue), syb2 + TMD-Cpx ΔCt21 (orange) or syb2 + TMD-Cpx ΔCt21-SBM (beige). In addition to the syb2 alone condition, equimolar TMD-anchored Cpx variants were also separately co-reconstituted into the v-SNARE vesicles. The plot shows the average percent of lipid mixing over time from five individual experiments that each contained three technical replicates. The standard error of the mean from each condition is shown as dotted lines. (c) Initial lipid mixing rates for each of the listed conditions, derived from the plots in panel b. (d) Total lipid mixing for each condition after 60 minutes, derived from the plots in panel b. **** denotes p-value < 0.0001; *** denotes p-value < 0.001; ** denotes p-value < 0.01; * denotes p-value < 0.05 and error bars represent standard error of the mean.

Source data

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Supplementary Video 1

Cpx creates pores and deforms GUVs. GUVs, labeled with rhodamine-PE (white) were generated with encapsulated recombinant HaloTag protein. The external media contains the fluorogenic HaloTag ligand, JF635i (Magenta). Application of 5 µM Cpx creates pores in the GUV membrane, allowing the JF635i dye to enter and bind the HaloTag protein, causing a fluorescence increase. Membrane remodeling and vesiculation is also observed. Scale bar represents 20 µm.

Source data

Source Data Fig. 1

Figs. 1b,e source data.

Source Data Fig. 2

Figs. 2e,f,g source data.

Source Data Fig. 5

Figs. 5e,g source data.

Source Data Fig. 6

Figs. 6b,d source data.

Source Data Extended Data Fig. 1

Extended Data Fig. 1a,b.

Source Data Extended Data Fig. 5

Extended Data Fig. 5c.

Source Data Extended Data Fig. 7

Extended Data Fig. 7c,d.

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Courtney, K.C., Wu, L., Mandal, T. et al. The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes. Nat Struct Mol Biol 29, 97–107 (2022). https://doi.org/10.1038/s41594-021-00716-0

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