The Role of Membrane Affinity and Binding Modes in Alpha-Synuclein Regulation of Vesicle Release and Trafficking

Abstract

Alpha-synuclein is a presynaptic protein linked to Parkinson's disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.

Keywords: Parkinson’s; alpha-synuclein; membrane; synapsin; synaptic vesicle.

Figure 1

Schematic illustrating the design logic for alpha-synuclein linker mutants. (A) Domain structure of alpha-synuclein and location of mutations used in the study. (B) Proposed functional contexts of different membrane-bound conformations of alpha-synuclein and the effects of the two linker region mutations, 4G and 3AE, on these conformations. The helix-1 and helix-2 regions are depicted as green rectangles and the linker region in the broken-helix state and the disordered C-terminal tail as orange lines. The linker region is highlighted with a red circle in both conformations, and the positions of the A30P and V70P mutations are marked with a star and a triangle, respectively. The two linker regions mutations are expected to inhibit conformational exchange and bias the membrane-bound conformation of the protein towards the broken-helix (4G) or extended-helix (3AE) state.

Figure 2

Effects of the linker region mutations on the micelle- and vesicle-bound states of alpha-synuclein. (A) NMR C-alpha secondary chemical shifts for micelle-bound WT alpha-synuclein and the 4G and 3AE mutants. Positive secondary shifts above ~1 PPM are indicative of significant helical propensity. Deuterated SDS concentration was 40 mM, and protein concentrations were 100–200 µM. Data were collected at 40 °C. (B) PRE in micelle-bound alpha-synuclein 4G and 3AE mutants labeled with a paramagnetic spin-label at position 9. SDS concentration was 40 mM, and protein concentrations were 100 µM. Data were collected at 40 °C. (C) Intensity ratios of signals from NMR ¹⁵N-¹H HSQC spectra of 50 µM alpha-synuclein variants obtained in the presence vs. the absence of SUVs at a total lipid concentration of 2.5 mM. Data were collected at 10 °C.

Figure 3

Functional assays and membrane affinity of alpha-synuclein variants. (A) Thapsigargin-stimulated exocytosis in RBL-2H3 cells transfected with low or high levels of WT or mutant alpha-synuclein measured using fluorescence of a VAMP8-pHlourin reporter. The difference in exocytosis levels between low and high expression levels represents the degree by which exocytosis is enhanced at high expression levels (t-test between high/low, p values *** < 0.001 < * < 0.05 < NS). (B) Lipid-binding curves for WT and mutant alpha-synuclein in an F4W mutant background measured by intrinsic tryptophan fluorescence as a function of increasing lipid concentrations. Protein concentrations were 0.1 µM, and lipid concentrations ranged from 1.25 µM to 10 mM. Data were acquired at a temperature of 22 °C (room temperature) by excitation at 280 nm and detection at 300–500 nm at 10 nm resolution, 60 flashes averaged and then baseline subtracted using a no-protein control, analyzed to extract bound fraction at every lipid/protein ratio and fit as described in the methods. Resulting fits are shown in solid lines. For each day of experiments with a new lipid vesicle preparation, WT data served as an internal control to account for variations. (C) Plot of membrane affinity derived from fits to the data in panel B vs. extent of enhancement of exocytosis derived from the data in panel A for WT and mutant alpha-synuclein.

Figure 4

Alpha-synuclein membrane binding profiles. DEST intensity ratios as a function of saturation offset and residue number for 100 µM WT (A), 3AE (B), V70P (C), 4G (D) and A30P (E) alpha-synuclein with 1 mM 60:25:15 DOPC/DOPE/DOPS lipid SUVs at 13 °C, using a 700 MHz spectrometer and a saturation bandwidth of 400 Hz. Broad profiles indicate exchange with a slowly tumbling membrane-bound state while narrow profiles indicate less or no membrane interaction.