In this study, we sought to investigate whether N-acetylation could modulate α-syn’s function in promoting SV clustering. Firstly, we prepared recombinant N-terminally acetylated α-syn (Ac-α-syn) as well as unmodified α-syn (un-α-syn). N-terminal acetylation leads to a significant chemical shift perturbation of the first nine residues and H50 of α-syn in the solution nuclear magnetic resonance (NMR) spectrum (Figure 1a, left panel). Intriguingly, in-cell NMR experiments show that all of the un-α-syn is N-terminally acetylated once it is delivered into HEK-293T cells by electroporation (Figure 1a, right panel), which is consistent with a previous report (Theillet et al., 2016). This result confirms the physiological relevance of N-terminal acetylation of α-syn. Notably, N-terminal acetylation is believed to be irreversible in mammalian cells due to the lack of a known N-terminal deacetyltransferase. Hence, it is crucial to investigate the impact of N-acetylation on the function of α-syn. To further probe the influence of N-acetylation on α-syn-mediated SV clustering, we incubated mouse SVs with various α-syn variants (Figure 1b, upper panel). We next measured the extent of SV clustering by utilizing dynamic light scattering (DLS) to monitor alterations in particle diameters, as well as using negatively stained transmission electron microscopy (TEM) (Figure 1b, middle and lower panel). Notably, N-acetylation significantly amplified α-syn’s capacity to cluster SVs, which requires the N-terminal thirty residues (Figure 1c–d). These findings imply that N-acetylation augments SV clustering.

In our previous work, we found that α-syn can bind to both negatively charged dioleoyl-phosphoserine (DOPS) (Diao et al., 2013) and neutral LPC (Lai et al., 2023). Building on this, we sought to examine how N-acetylation affects SV clustering that arises from the interaction between α-syn and either of these lipids. To assess clustering functionality with specific lipid compositions, we synthesized liposomes mimicking SVs that contained either LPC or DOPS. We then used a single-vesicle clustering assay to track the clustering mediated by Ac-α-syn and un-α-syn (Figure 2a). Importantly, we observed that N-acetylation considerably boosted the clustering ability of α-syn with LPC-containing liposomes (Figure 2b). In contrast, N-acetylation did not increase the clustering of liposomes containing DOPS (Figure 2c). Therefore, LPC is responsible for the increased SV clustering activity by N-terminally acetylated α-syn.

The interaction between α-syn and lipids is crucial for clustering SVs (Diao et al., 2013; Lai et al., 2023; Fusco et al., 2016). To elucidate the structural basis of how N-acetylation impacts the α-syn−LPC interaction, we carried out solution NMR titration analyses to examine the α-syn−LPC interaction with residue-specific resolution. Adding increasing concentrations of pure LPC micelles to unmodified α-syn resulted in a dose-dependent and continuous attenuation of signals for the first 100 residues of α-syn, indicating that these residues can interact with LPC (Figure 3a, upper). Notably, N-acetylation significantly elevated the binding affinity of the N-terminal region (residues 1–30), particularly the initial 10 residues, towards LPC, leading to a rapid binding pattern during titration (Figure 3a, lower). Moreover, Ac-α-syn demonstrated a sequential binding behavior with LPC, where the N-terminal region (residues 1–30) bound initially, succeeded by the central region (residues 30–100) as the LPC/Ac-α-syn ratio increased (Figure 3a, lower). Similar trends were observed in NMR titrations using DOPC/LPC-mixed liposomes (Figure 3b). On the other hand, N-acetylation only slightly weakened the interaction between the N-terminal region (residues 1–30) and DOPS, particularly at low DOPS/α-syn ratios, resulting in a continuous signal attenuation for the first 100 residues (Figure 3c). These findings reveal that N-acetylation considerably enhances the N-terminal region’s binding affinity, particularly the first 10 residues, to LPC, while modestly reducing the same region’s affinity to DOPS.

To provide insights that are more relevant to physiological conditions, we extended our investigation to examine how Ac-α-syn interacts with SV membranes. By utilizing a protocol established before (Wang et al., 2020), we prepared monodisperse SVs isolated from normal mouse brains and titrated them with Ac-α-syn at an SV/α-syn ratio of 260:3000, which is a physiologically relevant ratio within presynaptic terminals as determined in previous work (Wilhelm et al., 2014). The HSQC spectrum revealed signal attenuation, notably in the N-terminal region and specifically within the first ten residues of α-syn (Figure 3d). These findings closely mirrored those observed when Ac-α-syn was titrated with LPC micelles and DOPC/LPC-mixed liposomes at low lipid/α-syn ratios of 10:1 and 100:1, respectively (Figure 3e). These results further substantiate the critical role played by the N-terminal region of Ac-α-syn in SV membrane binding.

The intermolecular interactions between α-syn monomers (Fusco et al., 2016) or between α-syn and vesicle-associated membrane protein 2 (VAMP2) (Diao et al., 2013) are also essential for clustering SVs. To further characterize the accessibility of α-syn to LPC and DOPS, we performed cross-linking experiments coupled with mass spectrometry (XL-MS). We mixed ¹⁵N-labeled and unlabeled Ac-α-syn at a 1:1 ratio and selected the cross-linked fragments containing both ¹⁵N-labeled and unlabeled Ac-α-syn to map the intermolecular cross-linking pattern. We found the accessibility of Ac-α-syn to LPC to be much higher than that to DOPS (Figure 4a). The number of cross-linked peptide pairs, indicative of their close interaction, detected in LPC-induced multimers was over two-fold more than that of DOPS (Supplementary file 1a-f). This result demonstrates high intermolecular interactions between Ac-α-syn and LPC.

Lipids used in this study were purchased from Avanti Polar Lipids as follows: 16:0 LPC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) (855675), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (850375), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) (840035), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) (850457), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (850757), biotin-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)) (870277), cholesterol (70000).

N-terminal unmodified human α-syn (un-α-syn) and N-terminal 30-residue truncated α-syn (Δ30 α-syn) were cloned into the pET22 vector. For the N-terminal acetylated α-syn (Ac-α-syn), the fission yeast NatB complex (N-acetyltransferase) gene was co-expressed with α-syn in bacteria to acetylate its N-terminus (Johnson et al., 2010). ¹⁵N isotope-labeled Ac-α-syn or α-syn were produced in M9 minimal medium with uniformly labeled ¹⁵NH₄Cl (1 g/L, Cambridge Isotope Laboratories). Not isotope-labeled Ac-α-syn or α-syn were produced in the Luria-Bertani (LB) medium. Expression was induced by 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG) at 37 °C in E. coli strain BL21-DE3, which OD₆₀₀ reached 0.8–1.0. The collected bacteria were lysed in 100 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were boiled for 10 min followed by centrifugation at 16,000 g for 30 min. 20 mg/ml streptomycin was mixed with supernatants. After a 30 min centrifugation at 16,000 g, the pH value of supernatants was adjusted to 3.5 by 2 M HCl, followed by centrifugation at 16,000 g for 30 min. The supernatants were then dialyzed in 25 mM Tris-HCl (pH 8.0) overnight at 4 °C. The dialyzed protein solution was filtered through a 0.22 μm membrane (Merck Millipore, SLGP033RB) and injected onto an ion-exchange Q column (GE Healthcare, 17515601), followed by further purification via size exclusion chromatography (GE Healthcare, Superdex 75).

Neuronal SV were isolated from 8-week-old C57BL6 male mice (Lingchang Shanghai) by following published protocols (Ahmed et al., 2013). The experiment was performed at 4 °C or on ice with pre-cooled reagents. Four fresh brains (~0.5 g for each) were homogenized in 25 mL 4 mM HEPES-NaOH (pH 7.4) and 320 mM sucrose buffer (HB) with protease inhibitors by using a PTFE pestle in a 40 mL glass tube (Sigma, P7984). The homogenate was centrifuged at 1,500 g for 10 min. The supernatant (S1) was collected and kept on ice. The pellet was resuspended with 25 mL HB and homogenized, followed by centrifugation at 1500 g for 10 min. Then the supernatant (S2) combined with S1 was centrifuged at 20,000 g for 20 min. The pellet which contains synaptosomes was resuspended with 2 mL HB and then homogenized in 20 mL H₂O followed by adding 50 μL of 1 M HEPES-NaOH (pH 7.4) and protease inhibitors. The homogenate was placed on ice for 30 min, and centrifuged at 20,000 g for 20 min. The supernatant was ultra-centrifuged at 70,000 g for 45 min. The pellet was resuspended in buffers indicated in each analysis and homogenized by using a PTFE pestle in a 3 mL glass tube (Sigma, P7734). To further disrupt any remaining SV clusters before experiments, the homogenized SVs were drawn first through a 20-gauge hypodermic needle (Fisher Scientific, 0556121) attached to a 10 ml syringe, and then through a 27-gauge needle (Fisher Scientific, 22557176) and expelled. Mice experiments were conducted according to the protocols approved by the Animal Care Committee of the Interdisciplinary Research Center on Biology and Chemistry (IRCBC), Chinese Academy of Sciences (CAS).

The DLS experiments were performed using a DynaPro Nanostar instrument (Wyatt Technology, Santa Barbara, CA). A 50 μL aliquot of each sample containing homogenous SV (total protein concentration of 50 μg/mL) and α-syn variants (3 μM) was used for DLS measurement. The buffer used in this assay was 50 mM HEPES-KOH (pH 7.4), and 150 mM KCl. The following parameters were set for all measurements: correlation function low cutoff was 1.5 μs; correlation function high cutoff was 6×10⁴ μs; peak radius low cutoff was 0.5 nm; peak radius high cutoff was 10⁴ nm. The results were reported as the average of 10 consecutive autocorrelation functions (acquisition time = 5 s), and each sample was repeatedly measured three times.

An aliquot of 5 µL sample was pipetted onto a glow-discharged carbon-coated copper grid. 5 µL 3% (w/v) uranyl acetate was used for negative staining after washing the grid twice with 5 µL distilled H₂O. The grid was air-dried after removing extra uranyl acetate by filter paper. TEM images were obtained by an electron microscope (Thermo Fisher FEI Tecnai G2 TEM) at 120 kV equipped with a LaB₆ gun and a FEI BM Eagle CCD camera.

DOPS dissolved in chloroform was evaporated using a dry nitrogen stream. The dried lipid film was hydrated in buffers used for cross-linking (indicated below), and then sonicated in a water bath at 65 °C for 10 min. To mimic the size of synaptic vesicles, the hydrated lipids were extruded 41 times at 65 °C through a poly-carbonate film with a pore size of 50 nm (Whatman Nucleopore Track-Etch) by using an extruder apparatus (Avanti Polar Lipids, 610000). LPC powder was dissolved in buffers used for cross-linking for further analysis.

The protein-free liposomes (~100 nm) of POPC, POPE, cholesterol, LPC or DOPS, and biotin-DPPE or DiD (Invitrogen, D307) were prepared in the single-vesicle clustering buffer (25 mM HEPES-NaOH (pH 7.4), 100 mM NaCl) to a total lipid concentration of ~10 mM. The group of unlabeled liposomes contains POPC, POPE, cholesterol, LPC or DOPS, and biotin-DPPE with a molar ratio of (49.9~59.9):20:20:(0~10):0.1. The group of labeled liposomes contain POPC, POPE, cholesterol, LPC or DOPS and DiD with a molar ratio of (49~59):20:20:(0~10):1. LPC or DOPS on both vesicles was varied from 0 to 10%, in expense of POPC.

For the clustering experiments, the unlabeled liposomes (100 μM) were injected into the sample chamber and incubated for 30 min, followed by buffer exchange of 300 μL single-vesicle buffer. 10 μM DiD labeled liposomes were incubated with 20 nM α-syn or Ac-α-syn, and loaded into the sample chamber with 30 min incubation at room temperature. Unbound DiD liposomes and proteins were removed by buffer exchange of 300 μL single-vesicle buffer. Sample slides with multiple channels were monitored in a wide-field TIR fluorescence microscope. Data were acquired with an EMCCD camera, and analyzed with the smCamera program. Six images were taken at random locations within each channel on the quartz slide. The number of liposome interactions (clustering) was determined by counting the number of fluorescent spots from the emission of DiD upon excitation at 633 nm. Details were described previously (Diao et al., 2013; Diao et al., 2012).

Electroporation was performed with a Neon transfection system (Invitrogen, MPK5000). Lyophilized ¹⁵N-un-α-syn was dissolved in Buffer R (Invitrogen, MPK10025) to a final concentration of 300 μM. HEK-293T cells (ATCC, CRL-3216) were collected and washed with PBS for three times to get rid of the medium. Cells were resuspended in ¹⁵N-un-α-syn solution at 8×10⁷ cells per mL. Eight aliquots of 100 μL cell-protein mixture were pipetted and electroporated. Pulse program was: 1400 V pulse voltage, 20 ms pulse width, 1 pulse number. Electroporated cells were added into four 10 cm-diameter dishes with pre-warmed culture medium. Cells were allowed to recover for 5 hr, and were then harvested by trypsinization and washed with PBS four times. Resuspended cells were washed in 160 μL pH-stable L-15 medium (Gibco, 11415064) and 40 μL D₂O, and allowed to settle in a 5 mm NMR tube by gentle sedimentation with a hand-driven centrifuge. The supernatant was discarded. Finally, ~500 μL sedimented cell slurry was prepared for NMR measurement.

In-cell NMR experiments were carried out at 25 °C on a Bruker 900 MHz spectrometer equipped with a cryogenic probe by following a published paper (Wang et al., 2020). Bruker standard SOFAST-HMQC pulse sequence (Schanda and Brutscher, 2005; Schanda et al., 2005) was used and the ¹H shape pulse efficiency was optimized for collecting the 2D NMR spectrum of the in-cell samples with 80 scans. The delay time (D1) was set to 0.29 s, and 1024 and 128 complex points were used for ¹H and ¹⁵N, respectively. The experiment duration time is 61 min 16 s. Cell viability before and after the SOFAST-HMQC NMR experiment was assessed by trypan blue staining. Cell viability remained above 90% after the NMR experiments.

In vitro NMR experiments were carried out at 25 °C on an 800 MHz Agilent spectrometer equipped with a cryogenic probe (Agilent Technologies). The buffer used in all NMR titration assays was 50 mM sodium phosphate buffer (pH 6.5) containing 50 mM NaCl and 10% D₂O (v/v). All of the LPC or liposomes were prepared with a total molar concentration of 50 mM, and gradually added to the 500 μL 50 μM N-acetylated or unmodified α-syn in a series of NMR titration assays. In the NMR SV-titration experiment, each spectrum was acquired from separately prepared samples with increasing amounts of SV in 50 μM N-acetylated α-syn. The molar concentration of SVs was calculated according to parameters from a published paper (Takamori et al., 2006).

All the NMR data were processed using NMRPipe and analyzed by SPARKY (Delaglio et al., 1995). Backbone resonance assignment of N-acetylated and unmodified α-syn was accomplished according to a published paper (Theillet et al., 2016) and a Biological Magnetic Resonance Bank (BMRB) entry 6968 (Bermel et al., 2006), respectively.

LPC micelles, DOPS liposomes, and α-syn variants were mixed in a cross-linking-buffer (10 mM HEPES-NaOH, pH 7.4). The stock of cross-linker disuccinimidyl suberate (DSS) (Thermo Scientific, 21658) was dissolved in DMSO to 40 mM. 50 μM protein and 2 mM DSS were incubated at indicated concentrations of lipid in the cross-linking buffer at 25℃ for 1 hr. Crosslinking was terminated by the addition of 100 mM Tris-HCl (pH 7.4) at 25℃ for 10 min. For identification of the inter-molecular cross-linked peptide pairs from α-syn multimers by MS analysis, the half amount of α-syn was ¹⁵N-labeled α-syn.

The cross-linked samples were precipitated using acetone at −20 °C for 30 min. Precipitates were dried in air and resuspended in 8 M urea in 100 mM Tris-HCl (pH 8.5). Then, 5 mM TCEP was adding for 20 min incubation, followed by adding 10 mM iodoacetamide for 15 min for an alkylation reaction in the dark. Then, the samples were diluted to 2 M urea in 100 mM Tris-HCl (pH 8.5), and digested with trypsin (molar ratio of protein to trypsin was 50:1) at 37 °C for 16 hr in the presence of 1 mM CaCl₂ and 20 mM methylamine. Digestion was terminated by adding formic acid to 5% final concentration (v/v). After desalting by C18 desalting tips, the digested samples were air-dried and stored at –80℃ until analysis.

The digested peptides were analyzed by online nanoflow liquid chromatography-tandem mass spectrometry (LC−MS/MS). Briefly, nano LC−MS/MS experiments were performed on an EASY-nLC 1000 system (Thermo Scientific) connected to an Orbitrap Q Exactive HF (Thermo Scientific) through a nanoelectrospray ion source. The peptides were separated on a nano-column (C18, 100 μm×15 cm, 1.9 μm, 120 Å) and further analyzed using an Orbitrap Q Exactive HF mass spectrometer. A 60 min gradient was used in the nano LC at 300 nL/min flow rate: 0–4 min from 4% buffer B (80% ACN, 0.1% FA), 96% buffer A (0.1% FA in water) to 8% buffer B, 4–45 min from 8% buffer B to 22% buffer B, 45–53 min from 22% buffer B to 30% buffer B, then increasing to 95% buffer B in the next 41 min, and stay 95% buffer B for 3 min. One full-scan mass spectrum (350–1500 m/z) at a resolution of 60,000 followed by HCD fragmentation and detection of the fragment ions (scan range from 350 to 1500 m/z) in Orbitrap at a 27% normalized collision energy was repeated continuously.

The cross-linked peptide pairs were identified by pLink (Fan et al., 2015) first. The pLink search parameters used in this study include: (1) 20 ppm for a window of precursor mass tolerance. (2) 4 ppm for a window of fragment mass tolerance. (3) Lys for cross-linking sites of the cross-linker DSS. (4) 138.0680796 Da for xlink mass shift. (5) 156.0786442 Da for monolink mass shift. (6) 57.02146 Da for fixed carbamidomethyl-Cys modification mass shift. (7) 15.99491 Da for variable Met modification mass shift. (8) 4–100 amino acids per chain for peptide length. (9) 400–10,000 Da per chain for peptide mass. (9) 2 missed cleavage sites per chain for trypsin digestion of protein. The search results were filtered with a false rate of less than 5% and E-values of less than 0.0001.

The pLink readout data were filtered with a score less than 10^–6 and mass error for crosslinking assignments less than 4 ppm. To identify the inter-protein cross-linked peptide pairs, the spectrum (∆RT <0.5 s) of the two cross-linked peptide segments needs to be manually checked with four peaks of ¹⁴N-peptide and ¹⁴N-peptide, ¹⁴N-peptide and ¹⁵N-peptide, ¹⁵N-peptide and ¹⁴N-peptide and ¹⁵N-peptide and ¹⁵N-peptide.

HEK-293T cells were obtained from the American Type Culture Collection (CRL-3216).

C57BL6 male mice were from Lingchang Shanghai. All animal procedures were performed in accordance with the protocols approved by the Animal Care Committee of the Interdisciplinary Research Center on Biology and Chemistry, Chinese Academy of Sciences (mouse protocol number 20230110005).

All data are available in the main text or the extended data. Source data are provided in this paper. The XL-MS raw data and pLink searched data have been uploaded to iProX with the accession number IPX0009772000. Any data supporting the findings of this manuscript are available from the corresponding author upon reasonable request.