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Structural dynamics and transient lipid binding of synaptobrevin-2 tune SNARE assembly and membrane fusion - PubMed

  • ️Tue Jan 01 2019

Structural dynamics and transient lipid binding of synaptobrevin-2 tune SNARE assembly and membrane fusion

Nils-Alexander Lakomek et al. Proc Natl Acad Sci U S A. 2019.

Abstract

Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.

Keywords: NMR; SNARE proteins; dynamics; membrane fusion; neurotransmitter release.

Copyright © 2019 the Author(s). Published by PNAS.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

NMR analysis of syb-2. (A) Representative 2D 1H-15N TROSY-HSQC spectrum of 200 μM uniformly 2H,13C,15N-enriched syb-2 (1–96) dissolved in 20 mM MES buffer (pH 6.1), containing 250 mM NaCl, 1 mM EDTA, and 0.1 μM TCEP, recorded at 800-MHz magnetic field strength and 5 °C. (B) Schematic view of the domain structure of syb-2 (1–116). Residue numbers above the cylinder indicate the boundaries of the SNARE motif, the zero layer, the LD, and the TMR, respectively. (C) Cα − Cβ secondary chemical shifts (Δδ13Cα − Δδ13Cβ), plotted as a function of amino acid sequence number [values around zero (between −1 and 1) indicate random coil-like behavior, and positive values (>1) indicate α-helical propensity (around 3–4 for fully α-helical)]. Data reveal increased α-helical propensity for the C terminus of the SNARE motif and the N terminus of the adjacent LD. The strongly negative outlier for Val-8 can be explained by nearest neighbor effects due to Pro-9 (48).

Fig. 2.
Fig. 2.

Backbone dynamics of syb-2. The 15N relaxation data recorded for syb-2 (1–96) are shown. (A) 15N R1 relaxation rate constants recorded at 600 MHz versus residue. The red arrow highlights the unusually high values of 15N R1. The red line corresponds to the linear fit indicating the increase of rigidity of the protein backbone versus residue. (B) {1H}-15N NOE values versus residue increase linearly from the N terminus to the C terminus. The red line corresponds to the linear fitting indicating the increase of rigidity of the protein backbone versus residue. (C) 15N R2,0 relaxation rate constants recorded at 600 MHz (derived from 15N R1ρ with a 2-kHz RF field; R1 contribution-corrected) plotted versus residue. Red arrows highlight the two maxima in the dataset. (D) Hahn-echo transverse relaxation rates, R2β, at 800 MHz minus those measured at 600 MHz. Positive values indicate Rex contributions due to conformational dynamics on a microsecond-millisecond time scale. Regions subject to such conformational exchange processes are highlighted by red arrows.

Fig. 3.
Fig. 3.

Lipid interactions of syb-2 monitored by NMR. (A) Reference 1H-15N TROSY-HSQC spectrum of 200 μM 2H,13C,15N syb-2 (1–96) dissolved in 20 mM MES buffer (pH 6.1), containing 250 mM NaCl, 1 mM EDTA, and 0.1 mM TCEP, recorded at 800 MHz and 5 °C. Each [1H,15N] cross-peak corresponds to a visible (unbound) residue of syb-2 (1–96). (Side-chain amide signals of Gln are labeled by an asterisk. The spectrum is identical to the spectrum shown in Fig. 1A and is shown for comparison.) (B) Spectrum of 20 μM 2H,13C,15N syb-2 (1–116) (black) reconstituted into small liposomes constituted from a lipid mix of 5:2:2:1 DOPC/DOPS/DOPE/Chol in a buffer of 20 mM Hepes (pH 7.4 at 5 °C), 150 mM KCl, and 0.1 mM TCEP. The lipid/protein molar ratio was 100:1. For comparison, the spectrum is overlaid with the spectrum of syb-2 (1–96) (blue), identical to the spectrum shown in A. Signals from A74 to the C terminus are not visible in the spectra. (C) Spectrum of 20 μM 2H,13C,15N syb-2 (1–96) (black) in the presence of liposomes (5:2:2:1 DOPC/DOPS/DOPE/Chol), compared with the spectrum of syb-2 (1–96) without lipids (blue). In addition, soluble syb-2 signals from A74 to the C terminus disappear in the spectra, indicating lipid binding. Comparison is shown of normalized intensity ratios, Ilipid/Isolution, of the intensities in the 1H-15N TROSY-HSQC spectra for syb-2 (1–116) reconstituted in liposomes divided by the intensities of soluble syb-2 (1–96) (D), normalized intensity ratios of soluble syb-2 (1–96) in the presence of liposomes divided by soluble syb-2 (1–96) in the absence of liposomes (E), and normalized intensity ratio of syb-2 (1–116) reconstituted in liposomes divided by soluble syb-2 (1–96) in the presence of liposomes (F). (G) Percentage of lipid-bound syb-2 (1–116) population, as inferred from the intensity ratio shown in D via pbound=1−Ilipid/Isolution. (H) Same as in G, but for syb-2 (1–96). (I) Correlation plot between 15N R2 rate constants measured for syb-2 (1–96) and the percentage of bound population. Residues that show reduced mobility (inferred from higher R2 rate constants) in free syb-2 (without lipids) reveal a higher lipid binding affinity in the presence of liposomes. The Pearson correlation coefficient between the percentage of the bound population and R2 is ρ = 0.82.

Fig. 4.
Fig. 4.

Lipid binding of syb-2 monitored by fluorescence. Fluorescence spectra of NBD-labeled cysteine mutants were recorded between 500 nm and 640 nm in 20 mM Hepes, 150 mM KCl, and 0.1 mM TCEP (pH 7.4) at 8 °C. Blue traces show spectra acquired in the absence of liposomes, and black traces shown spectra acquired in the presence of liposomes. (Left Upper) Red trace shows spectra acquired in the presence of liposomes and unlabeled syb-2 in excess. The colored area represents the SD for n = 3.

Fig. 5.
Fig. 5.

Cooperative lipid binding of syb-2. Lipid binding of labeled syb-2 fragments after cleavage of syb-2 (1–96) with endoproteinase Glu-C. (A) Predicted cleavage sites (showing the residue numbers) of endoproteinase Glu-C from Staphylococcus aureus V8 in syb-2 (1–96) and electrostatic potential (determined using PyMOL, modified from Protein Data Bank ID code 2KOG). Orange circles indicate the labeled residue in each fragment. (B) Fluorescence spectra of NBD-labeled cysteine mutants 1C and K91C were recorded before and after digestion by endoproteinase Glu-C. Green traces show spectra acquired in the absence of liposomes, red traces show spectra acquired in the presence of liposomes before digestion, and blue and black traces show spectra acquired after digestion in the presence of liposomes (black) or without (blue). The colored area represents the SD for n = 3. (C) Hydrophobicity (ExPASy, ProtScale, Eisenberg et al.,

https://web.expasy.org/protscale/

) and charge (EMBOSS, charge,

www.bioinformatics.nl/cgi-bin/emboss/charge

) scores of syb-2 (1–96). A seven-residue window is used for both calculations. Dashed red boxes indicate the N-terminal and C-terminal fragments, respectively.

Fig. 6.
Fig. 6.

Lipid binding kinetics of syb-2. Stopped-flow measurements with an Alexa Fluor 488-labeled N-terminal cysteine mutant of syb-2. (A) Time course of Texas Red fluorescence emission at increasing total lipid concentrations is shown for syb-2 (lines) and at highest total lipid concentration for the SNARE complex (red symbols). (B) Observed kobs versus total lipid concentrations. The y-intercept provides the dissociation rate constant, koff, and the slope yields the association rate constant, kon. Bars represent the SEs of three to five technical repeats. The solid line shows a weighted linear fit (Eq. 2). (C) Fluorescence increase of Texas Red fluorescence emission is shown at different total lipid concentrations, in the absence (colored circles) and presence (black circles) of Ca2+, and fitted by a Hill equation (Eq. 3) with fixed n = 1. We obtained Kd = 602 ± 107 μM and Kd = 1,425 ± 119 μM, respectively. A.U., arbitrary unit.

Fig. 7.
Fig. 7.

Model of syb-2 dynamics and lipid binding in SNARE zippering and membrane fusion. In its precomplex state, syb-2 binds in a highly transient equilibrium to the vesicle membrane. Additionally, syb-2 shows decreasing flexibility from the N-terminal region to the C-terminal region that influences the rate of SNARE zippering. During exocytosis, intrinsic syb-2 membrane binding may help to compensate for repulsive forces between membranes and/or membranes and negatively charged side chains in the SNARE motifs, facilitating membrane fusion.

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