Solution NMR of SNAREs, complexin and α-synuclein in association with membrane-mimetics

Abstract

SNARE-mediated membrane fusion is a ubiquitous process responsible for intracellular vesicle trafficking, including membrane fusion in exocytosis that leads to hormone and neurotransmitter release. The proteins that facilitate this process are highly dynamic and adopt multiple conformations when they interact with other proteins and lipids as they form highly regulated molecular machines that operate on membranes. Solution NMR is an ideal method to capture high-resolution glimpses of the molecular transformations that take place when these proteins come together and work on membranes. Since solution NMR has limitations on the size of proteins and complexes that can be studied, lipid bilayer model membranes cannot be used in these approaches, so the relevant interactions are typically studied in various types of membrane-mimetics that are tractable by solution NMR methods. In this review we therefore first summarize different membrane-mimetic systems that are commonly used or that show promise for solution NMR studies of membrane-interacting proteins. We then summarize recent NMR studies on two SNARE proteins, syntaxin and synaptobrevin, and two related regulatory proteins, complexin and α-synuclein, and their interactions with membrane lipids. These studies provide a structural and dynamical framework for how these proteins might carry out their functions in the vicinity of lipid membranes. The common theme throughout these studies is that membrane interactions have major influences on the structural dynamics of these proteins that cannot be ignored when attempting to explain their functions in contemporary models of SNARE-mediated membrane fusion.

Figure 1

A schematic diagram of neuronal SNARE-mediated membrane fusion. Synaptobrevin on synaptic vesicles can bind to target SNAREs (syntaxin and SNAP-25) on the presynaptic cell membrane. The formation of the trans-SNARE complex (present in intermediate stage of diagram) may be modulated by various synaptic regulatory proteins, such as Munc18, Munc13, complexin, and α-synuclein. Fast and precise neurotransmitter release from the vesicle is triggered by the cytoplasmic rise of Ca²⁺ and its interaction with synaptotagmin. A four-helix bundle cis-SNARE complex is formed upon completion of fusion (final stage of diagram). Adapted with permission from reference [84].

Figure 2

Modular domain structures of neuronal SNAREs and related regulatory proteins. Rat protein sequences and isoforms are shown. The sequence lengths of these proteins are shown to scale, except that the scale of the last three proteins is five times smaller than the scale of the first five proteins. SNARE, SNARE motif; TM, transmembrane domain; N-pep, N-terminal peptide (residues 1-10); Habc, Habc regulatory domain of syntaxin (residues 27-146); CCCC, palmitoylated cysteines; NT, N-terminal (1-12); AH, accessory helix (28-47); CH, central helix (48-70); CT, C-terminal (114-134); NTR, N-terminal region (1-25); CR, central region (26-97); NAC, non-amyloid-β component (61-95); CTR, C-terminal region (98-140); CaMb, calmodulin binding region (459-492); MUN, MUN domain (859-1531).

Figure 3

Membrane mimetics for solution NMR studies of membrane proteins. (A) Micelle, (B) bicelle, (C) nanodisc, (D) amphipol, (E) Lipodisq or SMALP, (F) bilayer in a liposome. A hypothetical four-helical bundle protein is shown in green in all cases. Micelles and bicelles are shown in cut-open views in order to show the differences of these two systems. Membrane scaffold protein (MSP) helices in nanodiscs are shown in brown in (C); amphipols are shown in blue in (D); SMA polymers are shown as orange belts in (E).

Figure 4

Structural and dynamical changes of synpatobrevin in the presence of membrane lipids. (A–C) Three-bond averaged secondary chemical shift differences versus residue numbers, (A) synaptobrevin (1-96) in aqueous solution; (B) synaptobrevin (1-116) in DPC; (C) synaptobrevin (1-116) in DMPC/DHPC bicelles (q = 0.33). (D) Ratio of ¹⁵N R₂/R₁ relaxation rates for synaptobrevin (1-116) in DMPC/DHPC bicelles (q = 0.33). Adapted with permission from references [44, 46].

Figure 5

Structure models of synaptobrevin, syntaxin, complexin, and α-synuclein in lipid bilayer membranes. (A) Solution NMR structure of synaptobrevin in DPC (PDB code: 1KOG); (B) full-length syntaxin: solution NMR structure of syntaxin (residues 183-288) in DPC (PDB code: 2M8R) is linked with the NMR structure of the soluble Habc domain (residues 27-146) (1BR0) and the N-peptide (residues 1-12) (3C98); (C) complexin: the structure of the AH and CH helices, taken from the crystal structure of the complexin/SNARE co-complex (1KIL), is linked with the presumed helix-prone N- and C-termini that can tether complexin to the membrane; (D) solution NMR structure of α-synuclein in SDS micelles (1XQ8). The NTR and CTR can tether α-synuclein to the membrane. The protein domains are colored as in Figure 2.

Figure 6

Syntaxin (183-288) is well structured in DPC micelles. 1D-TRACT NMR experiments of (A) the full-length syntaxin (1-288), and (B) syntaxin (183-288) in DPC micelles. The best-fit single-exponentials to the Rα (red) and Rβ (blue) components are displayed as solid lines. (C) Two pairs of resonances showing different alignments in a 50% negatively charged acrylamide copolymer gel [85] and a final polymer concentration of 4%. (D) Observed H-N RDC values (red bars) versus residue numbers, with three stretches of helical segments (200-224, 227-247, and 264-283) fitted to dipolar waves, i.e., sinusoids of periodicity of ~3.6 residues [86]. Adapted with permission from reference [54].

Figure 7

N- and C-terminal regions of complexin both interact with membranes. (A) Overlaid HSQC spectra of complexin in buffer (10mM each HEPES, MES, and acetate, pH6, 150 mM NaCl, 1 mM EDTA) (red) and with the addition of 150 mM DPC (blue). Spectra were collected at 25 °C and 800 MHz. (B) Combined chemical shift changes Δδ_all (ppm), DPC-bound minus in buffer, versus their respective residue numbers. Here, Δδ_all is defined as [87]: $$\Delta {\delta }_{\mathit{\text{all}}}=\sqrt{{(0.154\ast \Delta {\delta }_N)}^2+{(0.276\ast \Delta {\delta }_{C\alpha })}^2+{(0.276\ast \Delta {\delta }_{C\beta })}^2+{(0.341\ast \Delta {\delta }_{CO})}^2}$$Each Δδ value on the right side of the equation is the chemical shift difference of that particular nucleus. ¹HN chemical shifts were not included in this calculation since ¹H chemical shifts are more sensitive to surrounding local magnetic fields, and hence the value Δδ_all reflects principally changes in backbone ϕ/ψ angles. (C) NMR intensity ratios of complexin bound to POPC nanodiscs (blue bars) or POPC/POPG (90/10) bilayers (red bars) relative to complexin in buffer. Adapted with permission from reference [59].

Figure 8

Solution NMR of α-synuclein. (A) Ratios of wild-type α-synuclein TROSY-HSQC peak heights in the presence (I) and absence (I_o) of lipid bilayer vesicles. Data from the following three samples are presented: N-terminally acetylated α-synuclein at a lipid:protein (L:P) ratio of 22 (red circles), nonacetylated α-synuclein at an L:P ratio of 22 (black triangles), and nonacetylated α-synuclein at an L:P ratio of 44 (black circles). Reproduced with permission from reference [75]. (B) In-cell HSQC NMR spectra of α-synuclein in A2780 and SK-N-SH cells (red) and of isolated N-terminally acetylated α-synuclein in buffer (black). AcM1, acetylated Met1. Reproduced with permission from reference [81].