Conformation of the synaptobrevin transmembrane domain

Abstract

The synaptic vesicle protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) forms part of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, which is essential for vesicle fusion. Additionally, the synaptobrevin transmembrane domain can promote lipid mixing independently of complex formation. Here, the conformation of the transmembrane domain was studied by using circular dichroism and attenuated total reflection Fourier-transform infrared spectroscopy. The synaptobrevin transmembrane domain has an alpha-helical structure that breaks in the juxtamembrane region, leaving the cytoplasmic domain unstructured. In phospholipid bilayers, infrared dichroism data indicate that the transmembrane domain adopts a 36 degrees angle with respect to the membrane normal, similar to that reported for viral fusion peptides. A conserved aromatic/basic motif in the juxtamembrane region may be causing this relatively high insertion angle.

Fig. 1.

CD experiments. (A) CD spectra of synaptobrevin constructs in detergent. Full-length synaptobrevin is blue, the cytoplasmic domain is green, and the transmembrane peptide is red. The data are plotted as molar ellipticity [θ]_ME (×10⁻³ °·cm⁻²·dmol⁻¹) because of the different lengths of the constructs. All spectra were taken at a 100 μM protein concentration in 50 mM βOG. (B) Cartoon illustrating the constructs used in A. The full-length (FL) contained residues 1–116, the cytoplasmic construct (CYT) contained residues 1–96, and the transmembrane construct (TM) contained residues 74–116. The labels are colored according to line colors in A. (C) Sensitivity of secondary structure to detergent for the transmembrane construct at a concentration of 100 μM. The detergent concentrations are five times the reported critical micelle concentrations. The CD spectra were measured in the presence of 13 mM SDS (red), 48 mM cholate (blue), and 95 mM βOG (green). DMPC (1.5 mM) is shown in black. The data are plotted as mean residue ellipticity [θ]_MRE (×10⁻³ °·cm⁻²·dmol⁻¹ per residue). (D) Effect of increasing of TFE concentration on secondary structure in the transmembrane domain. The transmembrane construct is present at 100 μM with 50 mM βOG. Separate samples were prepared for each spectrum. The CD spectra of the synaptobrevin transmembrane construct are shown without TFE (black), 5% TFE (red), 10% TFE (green), and 30% TFE (blue). The data are plotted as [θ]_MRE.

Fig. 2.

IR absorbance experiments. (A) IR absorbance spectra of the amide I region of synaptobrevin transmembrane constructs in POPC bilayers. The labeled constructs contain one leucine residue with ¹³C at the indicated carbonyl carbon, L84 (green), L93 (red), L99 (magenta), and L107 (blue). The ¹³C-shifted side peaks are indicated with arrows. (B) Sequence of the synaptobrevin transmembrane construct depicting positions of the labeled ¹³C leucine residues. The numbering is for rat synaptobrevin 2 (NP_036795). The position of the predicted transition between transmembrane helix and unstructured region is indicated by the star.

Fig. 3.

Representative polarized IR absorbance spectra used to generate order parameters for the tilt angle calculation. The black curve represents absorption of parallel (0°) polarized light. The gray curve represents the absorption of perpendicular (90°) polarized light. The ratio of these absorbances is used to determine R^ATR. (A) Region of the spectrum corresponding to the lipid acyl-chains for the L99-labeled construct in DMPC. (B) Region of the spectrum corresponding to the protein amide I for the same construct. (C) Region of the spectrum corresponding to the lipid acyl-chains for the L84 labeled construct in POPC. (D) Region of the spectrum corresponding to the protein amide I for the same construct.

Fig. 4.

Model of synaptobrevin in a phospholipid bilayer. The transmembrane peptide construct is shown in blue as an idealized α-helix with the break in helical structure predicted from CD and IR spectra. Tryptophan and lysine side chains are shown in stick representation and are displayed in the most favored rotamer positions. A simulated DMPC bilayer is shown for illustration of scale. Bilayer carbon atoms are purple, oxygen atoms are red, and phosphorous atoms are green. The transmembrane domain was placed at a 35° tilt relative to the bilayer normal. The vertical position of the peptide was set by placing the tryptophan residues near the interfacial region of the membrane (22, 59, 62). The model was prepared with pymol (Delano Scientific, San Carlos, CA). (The coordinates for the bilayer were obtained from the Department of Biocomputing at the University of Calgary, Calgary, AB, Canada.)

Fig. 5.

Alignment of synaptobrevin juxtamembrane region and transmembrane domain. The numbering above the alignment is that of rat synaptobrevin 2. The alignment was performed with clustalw with the PAM 350 matrix (66). The Medline unique identifier is indicated before each sequence followed by the isoform and species information. The image was prepared with espript (67). Residues that are >70% conserved are colored red and highlighted with a blue box.