1. Volume 113, Issue 6, Pages 1235-1250 (September 2017)
Complexin Binding to Membranes and Acceptor t-SNAREs Explains Its Clamping Effect on Fusion 
Rafal Zdanowicz, Alex Kreutzberger, Binyong Liang, Volker Kiessling, Lukas K. Tamm, David S. Cafiso 
Biophysical Journal 
Volume 113, Issue 6, Pages (September 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Complexin interacts with the core SNARE complex. (a) Complexin-1 contains regions reported to bind membranes and SNAREs. The crystal structure of a fragment of Cpx bound to the core SNARE complex is shown (PDB: 1KIL) where SNAP-25a, syntaxin-1a, and synaptobrevin-2 helices are indicated in green, red, and blue, respectively. In this study, the spin-labeled side chain R1 was engineered into multiple sites in Cpx, covering the N- and C termini, and the central helical region (magenta spheres indicate the position of several labeled sites on Cpx in the Accessory and Central helices). (b) Shown here are X-band cw-EPR spectra for 13 spin-labeled Cpx mutants in solution (black trace) or incubated with the assembled core SNARE complex (red trace). Sites that interact with the SNAREs exhibit a broadening of the EPR spectrum and a decrease in normalized amplitude. All spectra are 100 Gauss scans with at least 10 averages taken. (c) Shown here is the ratio of the normalized intensities for the high-field resonance line of each mutant in the presence/absence of the core SNARE complex. Error bars represent the uncertainty in the normalized intensity ratio due to normalization and measurement of peak-to-peak amplitudes. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
Complexin binds to soluble binary t-SNARE complex. (a) Shown here is the 15N-1H HSQC spectrum of Cpx (26–83) in buffer. Assignments of backbone amides are denoted by one-letter amino acid abbreviations followed by their sequence numbers. (b) Shown here is the 15N-1H HSQC spectrum of Cpx (26–83) mixed with an equimolar concentration of sBC, consisting of Syx(191–253) and SNAP25 (green), overlayed onto the HSQC spectrum of free Cpx (26–83) (red). (c) Shown here are intensity ratios of backbone amide resonances versus residue numbers at different molar ratios of Cpx (26–83)/sBC, with ratios of 10:1, 5:1, and 1:1 represented in red, blue, and green bars, respectively. The intensity ratios are the intensities of the Cpx (26–83)/sBC samples over the intensities of Cpx (26–83) alone and were calibrated using N- and C-terminal residues to account for differences in sample concentrations. Only peaks with S/N higher than 20 were analyzed, and error bars were propagated from S/N levels of spectra and calibrated with redundant measurements. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Complexin binds to membranes at both its N- and C-terminal regions. (a) Shown here are NMR intensity ratios of Cpx bound to POPC nanodiscs (cyan bars) or POPC/POPG (90:10) bilayers (red bars). (b) Shown here are EPR spectra in the absence (black trace) and presence (red trace) of POPC/POPS (70:30) liposomes and (c) intensity ratios for the high field resonance of spin-labeled Cpx mutants with/without POPC/POPS (70:30) liposomes. Error bars represent the uncertainty in the normalized intensity ratio due to normalization and measurement of peak-to-peak amplitudes. The data indicate the N- and C termini of Cpx are involved in direct membrane binding because the largest intensity drops in both the NMR data (a) and EPR data (b) are seen for the N- and C-terminal regions. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
Complexin senses membrane curvature. (a) EPR spectra of E2R1 and T119R1 show dramatic changes in lineshape upon membrane binding, and these changes were used to determine Cpx membrane affinity. Titrations of (b) E2R1 and (c) T119R1 with unilamellar vesicles of varied size (≈100, ≈50, and ≈25 nm in diameter) yield the fraction of membrane bound Cpx as a function of accessible lipid. These data were fit (solid lines) to a simple binding isotherm to yield the membrane binding partition coefficient shown in Table 2 (see Materials and Methods). In (d), the binding of Cpx119R1 in full-length Cpx was compared with that for an N-terminally truncated version Cpx (26–134) to small 25-nm-diameter vesicles, and indicate that the N- and C-terminal regions act independently. All data points are averages of three titrations. Error bars represent deviations from the best fit. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Membranes with the reconstituted acceptor t-SNARE complex enhance the membrane binding of Cpx. Shown here are EPR spectra from different spin-labeled Cpx mutants in solution (black traces) or incubated with either (a) liposomes at 4 mM lipid (70:30 POPC/POPS) (cyan trace) or (b) proteoliposomes with 4 mM lipid and t-SNARE complex (70:30 POPC/POPS) at a protein/lipid ratio of (1:100) (red trace). (c) Shown here are normalized intensity ratios for spin-labeled Cpx in the presence/absence of liposomes (4 mM lipid) (cyan bars) or in the presence/absence of proteoliposomes with t-SNAREs (red bars). The ratios are determined from normalized intensities measured from the high-field nitroxide resonance line (MI = −1). The error range represents the uncertainty in the determination of the ratio. Protein-free liposomes were prepared by dialysis in an identical manner to that for the proteoliposomes. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Complexin strongly binds to proteoliposomes with reconstituted acceptor t-SNARE complexes. (a) Shown here are binding curves for Cpx, using the spin-labeled mutant CpxT119R1, produced by recording EPR spectra with increasing concentrations of proteoliposomes that were either protein-free (black trace) or reconstituted with Syx (red trace), dSNAP25 (blue trace), the Syx:dSNAP25 t-SNARE complex (green trace), or the assembled cis-SNARE complex (orange trace). All proteins were present at a protein/lipid ratio of 1:100. Points are averages of three titrations, and errors represent deviations from the best fit. (b) Fluorescence anisotropy measurements are consistent with EPR, where fluorophore-labeled Cpx was mixed with either protein-free liposomes, dSNAP25, Syx, or t-SNARE-containing proteoliposomes composed of 70:30 POPC/POPS at a lipid/protein ratio of 400:1. The effect of the interaction was quantified and plotted as a change in fluorescence anisotropy relative to free Cpx. Anisotropy changes are the average of four experiments, and the error bars represent standard errors. (c) The binding of Cpx to planar-supported bilayers was measured with TIRF microscopy. Increasing concentrations of labeled Cpx were titrated into the planar bilayer, and increases in fluorescence in the TIRF field were monitored. The fluorescence as a function of Cpx concentration is shown for protein-free planar bilayers (70:30 POPC/POPS) (red trace) and for planar bilayers (70:30 POPC/POPS) reconstituted with syntaxin-1/dSNAP25 (lipid/protein of 3000) (black traces). For each condition, values are averages from three separate bilayer preparations. Error bars are standard errors. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
Complexin inhibits membrane fusion by reducing synaptobrevin affinity for t-SNAREs. (a) Shown here is lipid mixing between Syb and binary acceptor t-SNARE complex proteoliposomes (t-SNARE membrane composed of 70:30 POPC/POPS with a lipid/protein ratio of 400; v-SNARE membrane composed of 97:1.5:1.5 POPC/Rh-DOPE/NBD-DOPE, lipid/protein ratio of 400). Observed fluorescence is a function of time, and presented as normalized signal intensity. After each fusion reaction, 0.1% Triton X-100 was added to determine total fluorescence and normalize the data between different preparations. Traces correspond to lipid mixing in the absence of Cpx (black) or in the presence of 1 (red), 2 (blue), 4 (violet), or 8 μM Cpx (green trace), as indicated. All traces are normalized to 0 μM complexin. (b) Saturation of the lipid mixing taken at 500 s as a function of the concentration of added Cpx. The inhibitory effect of Cpx is concentration dependent, and at 8 μM, Cpx has reduced fusion by ∼20-fold. Error bars are standard errors from four measurements. (c) Shown here is docking of synaptobrevin proteoliposomes to SNARE acceptor complexes reconstituted into planar-supported bilayers (Syb proteoliposomes composed of 99:1 POPC/Rh-DOPE) as a function of time. The docking was measured in the absence of Cpx (black) and in the presence of 0.5 μM (red), 1 μM (cyan), and 2 μM Cpx (green), as indicated. (d) The number of bound Syb proteoliposomes at 1200 s as a function of concentration of Cpx. Values are averages from measurements on three bilayers, and errors are standard errors. (e) Shown here is the binding of a fluorescently labeled soluble Syb (1–96) peptide after a 20 min incubation to a planar-supported bilayer (70:30 POPC/POPS) containing the t-SNARE complex (SyxH3/dSNAP25) at a lipid/protein ratio of The binding of Syb was measured without Cpx (black), and at Cpx concentrations of 0.5 μM (red), 1 μM (blue), and 2 μM (green). Values are averages of three measurements and errors are standard errors. (f) The Kd for Syb binding calculated from the data in (e) is shown as a function of Cpx concentration for wild-type Cpx (black trace) as well as the superclamping (SC, blue trace) and nonclamping mutants (NC, purple trace). Error bars represent error in the fit. The mutations of SC are D27L, E34F, and R37A (42), and the mutations of NC are A30E, A31E, L41E, and A44E (32). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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