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1. Saturation: CEST starts by selectively saturating (or exciting with a strong radiofrequency pulse) the protons of interest that are bound to molecules other than water (could be protons in metabolic products or proteins)

2. Chemical Exchange: Following saturation, these protons exchange with the protons in water molecules

3. Detection: When the water protons (which are now in excess and not initially targeted by the saturation pulse) exchange with the saturated protons, they carry some of the saturation with them, this exchange alters the signal from the water protons that is detected by the MRI

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Measures Rex: The contribution of chemical exchange to the transverse (R2) relaxation rate

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Low R₂/R₁ ratio --> small R2 and large R1 --> rigid, compact protein or region

Large R₂/R₁ ratio --> large R2 and small R1 --> flexible, non-compact protein or region

Changes in R1 and R2 rates upon binding to ligands or other proteins can indicate the interaction interfaces and conformational changes e.g. decrease in R₂/R₁ ratio upon saturation with ligand implies protein becomes more compact upon binding

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¹HN --> ¹⁵N --> ¹³Cα --> ¹³CO

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¹HN --> ¹⁵N --> ¹³CO

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CBCANH/HNCACB, CBCA(CO)NH/HN(CO)CACB and HSQC form the standard set of experiments needed for backbone assignment

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Data are processed with two FT across the indirect and direct dimensions

1. FT t₂ (directly-detected dimension in the ¹H plane) --> peak at same point on y axis (corresponding to the resonant frequency of the nucleus being observed) but with modulated intensity due to the influence of indirect evolution in the ¹⁵N plane (t₁ dimension)

2. FT across t₁ (indirectly-detected dimension in the ¹⁵N plane) --> single peak in the resulting 2D spectrum

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Protein dynamics are directly related to function

They take place at a range of timescales that are relevant to biology

NMR is the prime method to study protein motion

ns-ps dynamics: Motion within an energy well --> T1, T2 relaxation, het NOE

μs-ms dynamics: Exchange between different states --> CPMG and CEST/DEST

IDPs are a classic example of functionally relevant dynamics: Chemical shifts (local structure) and PREs (topology)

ssNMR can be used to study large assemblies

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CEST provides key information from indirect measurements

Wide range of applications: from protein-ligand binding to protein-membrane interactions, and medical applications in MRI

αS function and aggregation can be characterised by a number of solution and ssNMR methods

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GluCEST MRI: Exchange of bulk water and amine protons of glutamate 

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GluCEST MRI: Exchange of bulk water and amine protons of glutamate 

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Glutamate (Glu), a neurotransmitter in the brain, shows a pH- and concentration-dependent chemical exchange transfer effect (GluCEST) between its amine group and bulk water

GluCEST map shows the regional distribution of Glu in the brain, higher GluCEST contrast observed in gray matter compared to white matter

Sensitive to [Glu] and pH

Contrast favourable at high fields (≥ 7 T)

Imaging of: Stroke, Alzheimer's, Huntington's e.g. AD mice exhibited notable reduction of GluCEST contrast (~30%) in all areas of the brain

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Practical data acquisition method for human brain tumor amide proton transfer (APT) imaging

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Practical data acquisition method for human brain tumor amide proton transfer (APT) imaging

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Chemical exchange saturation transfer (CEST): What is in a name and what isn't?

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Chemical exchange saturation transfer (CEST): What is in a name and what isn't?

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CEST imaging is a relatively new MRI technique in which exogenous or endogenous compounds containing either exchangeable protons or molecules are selectively saturated and after transfer of this saturation detected indirectly through the water signal with enhanced intensity

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Exchangeable protons that resonate at a different frequency from the bulk water protons

The bulk water signal (4.75 ppm) is saturated when we use a saturation band applied at 8.25 ppm (saturates amides??)

Saturate solute protons--> transfer of saturated protons to water --> water signal goes down

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CEST MRI: Exchange of bulk water and amide protons 

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CEST MRI: Exchange of bulk water and amide protons 

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Allows easy discrimination of tumors (and grade), neurodegeneration etc.

Sensitive to acidosis (pH modification)

Applicable at clinical fields (≤ 3 T)

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Opportunities for NMR to study membrane protein interactions

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Opportunities for NMR to study membrane protein interactions

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ssNMR in the cross polarisation regime for rigid regions of membrane bound proteins

ssNMR in the INEPT region for dynamic regions of membrane-bound proteins

CEST probing the interaction from the unbound state perspective

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Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers

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Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers

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Non-toxic and toxic structures, differences in terms of 2o structure

Toxic oligomer structure has more dynamic N terminus

IDPs can form many different structures even when in oligomers

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Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour

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Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour

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Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour - Nature Communications

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α-synuclein is a protein whose aberrant aggregation is associated with Parkinson’s disease. Here, Fusco et al.characterize α-synuclein bound to lipid membranes using a combination of solution and solid-state NMR spectroscopy and provide insights into the molecular processes associated with the aggregation of this protein.

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Amyloid fibrils good cryo EM structural studies

Highly toxic oligomers much harder to study structurally (transient, unstable)

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C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

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C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

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C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction - Nature Communications

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Alpha-synuclein is associated with neuronal dysfunction in Parkinson’s disease. This study shows that alpha-synuclein interacts with neuronal synaptic vesicles in a calcium-dependent fashion, and this interaction is important for synaptic vesicle clustering.

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E46K/K80E mutant has enhanced vesicle fusion activity

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E46K/K80E mutant has enhanced vesicle fusion activity

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K80E --> more negative --> binds 2nd membrane better --> promotes vesicle fusion

Primary requisites: Same aminoacidic composition, same overall charge

Intended requisites: Same membrane affinity, more exposed NAC region

Modulated by Ca²⁺

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αS brings together two different lipid membranes, may promote vesicle fusion

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We can probe the binding of αS to membranes on a per-residue basis

Determine the regions that are bound and the kinetics of binding

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Structural basis of synaptic vesicle assembly promoted by α-synuclein

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Structural basis of synaptic vesicle assembly promoted by α-synuclein

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α-Synuclein (αS) is a 140-residue protein whose aggregation has been strongly associated with Parkinson's disease (PD)1,2,3,4,5. Fibrillar deposits of αS are the major constituents of Lewy bodies6,7,8, a hallmark of the disease, and inherited forms of early onset PD are associated with mutations, duplications and triplications of the αS-encoding gene9,10. Despite the general consensus on its pathological relevance, the physiological role of αS remains widely debated. In this context, a view is emerging in which αS is involved in the dynamics of synaptic vesicle (SV) trafficking by regulating a distal reserve pool of SVs that controls the amount of vesicles docked at the synapses during neurotransmitter release11,12. This biological role is directly associated with the ability of αS to bind to synaptic vesicles and induce their interaction and assembly in vitro and in vivo 13,14,15,16. Indeed, in dopaminergic neurons αS exists in a tightly regulated equilibrium17 between a cytosolic monomeric form, which is predominantly disordered18,19,20, and a membrane-bound state, which is rich in α-helix structure in the region spanning residue 1-90 of the protein sequence15,21,22,23,24,25,26.

Because of its intrinsic ability to bind to a wide variety of biological membranes, the physiological state of membrane-bound αS is extremely difficult to characterize, as a variety of factors, including the presence of detergents22 and chemical modification of the protein27, can alter dramatically the structural properties of its bound state15. In a recent study, three major regions were identified to have distinct structural and dynamical properties that influence in different ways the nature of the membrane-bound state of αS28; these regions include an N-terminal α-helical segment, acting as the membrane-anchor, an unstructured C-terminal region, weakly associated with the membrane, and a central region, undergoing order-disorder transitions in the membrane-bound state and determining the affinity of αS for lipid bilayers of different composition28. This structural variability indicates that it is of fundamental importance to investigate the binding of αS to lipid membranes under conditions that reproduce as closely as possible the physiological environment relevant to that of presynaptic vesicles15.

We describe here a detailed characterization of the dynamical and structural properties at the surface of synaptic-like lipid vesicles of two familial αS mutations that have opposite effects on its affinity for membrane binding29,30. On the basis of these studies, we characterized the details of the underlying mechanism by which a single molecule of αS binds two different synaptic vesicles and promotes their interaction and assembly. This mechanism, which involves a double-anchoring step enabling αS to form a dynamic link between two vesicles, is strongly supported by an experiment in which a variant of αS was engineered to adopt structural properties in its membrane-bound state that result in enhanced αS-mediated interactions between synaptic vesicles while maintaining the same amino acid composition, charge and membrane-binding affinity of the wild-type protein. The mechanism, which was verified using both synthetic lipid vesicles and synaptic vesicles purified from rat brain, provides evidence that the specific level of affinity for membrane binding of the non-amyloid-β component (NAC) region of αS is a fundamental functional property enabling this protein to mediate the interaction between vesicles.

Binding of αS variants to membranes

Using solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with cryo-electron microscopy (cryo-EM) and stimulated emission depletion (STED) imaging, we have characterized the structural properties at the surface of synaptic-like vesicles of the familial αS mutants A30P31 (αS A30P) and E46K32 (αS E46K) and compared their behaviour with that of the wild-type protein (αS WT)28. In particular, we studied the interactions of αS A30P, αS E46K and αS WT with small unilamellar vesicles (SUVs) composed of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in 5:3:2 molar ratios29, as such SUVs have been reported to be good mimics of synaptic vesicles for composition and curvature15. The combination of magic angle spinning (MAS33,34) measurements in solid-state NMR (ssNMR35) and chemical exchange saturation transfer (CEST28,36,37,38,39,40, Supplementary Fig. 1) experiments in solution-state NMR used in this study has already been shown to be highly effective in probing interactions between αS and SUVs, enabling the degree of order and disorder in the membrane-bound state of αS WT to be characterized28.

In the analysis of the interaction between αS A30P and SUVs, CEST profiles ( Fig. 1 and Supplementary Fig. 2) provided detailed information concerning the effects of the A30P mutation, which was shown to reduce very substantially the binding affinity of αS for SUVs15. In αS A30P, indeed, the binding of the N-terminal anchor region was found to involve a smaller number of residues than in the case of αS WT (the N-terminal 20 residues compared with the N-terminal 25 residues in αS WT) with generally a lower degree of CEST saturation than in the case of αS WT. The major differences between αS A30P and αS WT were evident in this membrane-anchor N-terminal region, while the remainder of the protein sequence showed very limited variations in the CEST profiles of these two proteins. The opposite behaviour was observed for the αS E46K variant, which binds SUVs with higher affinity than does αS WT 15. Indeed, the CEST data indicate a significantly stronger interaction with the membrane for the N-terminal anchor region of αS E46K, which in this case extends up to residue 42 with a generally higher degree of saturation than in the case of αS WT. As with αS A30P, only marginal variations in the CEST profiles were observed in other regions of the sequence of αS E46K. The differences in the CEST saturation profiles of the two mutants compared with αS WT ( Supplementary Figs 3 and 4) indicate more specifically that the major changes in the modes of binding to SUVs of these mutational variants are associated primarily with the N-terminal region of the protein.

CEST experiments were recorded at a ¹H frequency of 700 MHz (see Methods section), using protein concentrations of 300 μM and 0.06% (0.6 mg ml −1) of DOPE:DOPS:DOPC lipids in a ratio of 5:3:2 and assembled into SUVs. ¹H- ¹⁵N HSQC spectra were recorded by using continuous wave saturation (170 Hz or 350 Hz) in the ¹⁵N channel at offsets ranging between −28 kHz and +28 kHz; an additional spectrum, saturated at −100 kHz, was recorded as a reference. Data recorded using a saturation bandwidth of 350 Hz are shown here (the data measured using a saturation bandwidth of 170 Hz are shown in Supplementary Fig. 2). For comparison, the plots in panels b and e are drawn using αS WT data from our previous investigation28. ( a- c) CEST surfaces for αS A30P ( a) αS WT 28 ( b) and αS E46K ( c). ( d- f) CEST saturation along the sequences of αS A30P ( d), αS WT 28 ( e) and αS E46K ( f). The green lines refer to the averaged CEST profiles measured using offsets at +/− 1.5 kHz, and the profiles for +/− 3 kHz and +/− 5 kHz are shown in black and red, respectively. ( g- i) Schematic illustration (see Materials and Methods) of the equilibrium between surface attached/detached local conformations in the membrane-bound states αS A30P ( g) αS WT 28 ( h) and αS E46K ( i). The major differences in the data of αS A30P, αS WT and αS E46K are located in the anchor region. Overall, these three variants of αS maintain the same topological properties at the surfaces of synaptic-like SUVs.

Topology of αSA30P and αSE46K bound to SUVs

To obtain detailed information on the topology of αS A30P and αS E46K when bound to the surface of SUVs, we used MAS ssNMR experiments. ¹³C- ¹⁵N-labelled αS samples were mixed with SUVs, as described previously,15,28 to reach a protein:lipid ratio of 1:65 (ref. 28). Under these conditions we could observe directly the resonances of both rigid and dynamical regions of the membrane-bound αS molecule by using cross polarization and insensitive nuclei enhanced by polarization transfer (INEPT) experiments41, respectively. In the cross polarization regime, we performed ¹³C- ¹³C dipolar-assisted rotational resonance (DARR)42 measurements to detect homonuclear correlations between carbon atoms of residues strongly anchored to the membrane ( Fig. 2a). In our previous study of αS WT 28, the ¹³C- ¹³C DARR spectra identified resonances of residues 6-25 of the anchor region, showing that this region folds into a highly rigid α-helix lying essentially parallel to the membrane surface28. The ¹³C- ¹³C DARR spectra of the membrane-bound states of αS A30P and αS E46K are, however, substantially different from those of αS WT, indicating that the dynamical and structural properties of the anchor region vary considerably between the wild-type and variant forms of αS.

( a) ¹³C- ¹³C DARR correlation spectra (aliphatic regions) recorded at −19 °C using a 50 ms mixing time at a MAS rate of 10 kHz. We used a 1:65 protein:lipid ratio in both cases, and spectra of αS A30P and αS E46K are shown in the left and right panels, respectively. Residues are indicated using the single letter convention. The highest signal intensities in the spectra of the samples studied here were obtained by performing the measurements at −19 °C. Under these conditions the lipid mixtures used here are in the gel phase70, enabling ¹³C- ¹³C DARR spectra to be measured with significantly increased signal-to-noise ratios but without affecting the pattern of chemical shifts; the latter are consistent with those measured at 4 °C (ref. 28). No variations in the number of observed resonances or in the chemical shifts were observed using protein:lipid ratios ranging from 1:30 to 1:200 (ref. 28). ( b) ¹H- ¹³C correlation via INEPT transfer recorded at 4 °C at a MAS rate of 10 kHz. The experiments were performed at a ¹H frequency of 700 MHz using a 3.2 mm E Free probe. Atom names ca, cb, cg, cd and ce are used for C α, C β, C γ, C δ and C ɛ atoms, respectively.

In the case of αS A30P, the ¹³C- ¹³C DARR spectrum showed a very limited signal-to-noise ratio and almost a complete absence of cross peaks, suggesting that the anchor region of this variant is significantly more dynamic than the same region of the wild-type protein. In contrast, the ¹³C- ¹³C DARR spectrum of αS E46K showed a higher signal-to-noise ratio and a significantly larger number of intense cross peaks, indicating an elongated anchor region in this mutational variant that binds more strongly to SUVs. Using the dipolar connectivities from ¹⁵N- ¹³C cross polarization-based experiments, along with our previous assignment of αS WT and ¹³C- ¹³C DARR spectra acquired at different mixing times, we were able to assign individual spin systems in the ¹³C- ¹³C DARR spectra of αS A30P and αS E46K ( Fig. 2a). The chemical shifts were then compared with those obtained from solution-state NMR studies of αS WT in SDS and SLAS micelles22,43, and indicate that, despite the differences in dynamics and in the binding strength relative to lipid membranes, all the variants analysed here adopt a helical conformation at the N-terminal anchor when bound to membranes. This finding is particularly relevant in the case of the A30P mutation as it shows that, like the other variants and despite the insertion of a helix-breaker residue, αS A30P binds the lipid bilayer by adopting an amphipathic α-helix conformation at the N terminus and not as a disordered state that is tethered onto the lipid surface.

The highly dynamical regions of αS A30P and αS E46K bound to SUVs were then probed directly by INEPT measurements acquired using MAS ssNMR experiments41 and revealed ¹H- ¹³C correlations for resonances of the disordered C-terminal region of αS ( Fig. 2b), which is only weakly associated with the membrane28. In contrast to the results obtained from the DARR spectra, no significant differences were found between αS A30P and αS E46K in the INEPT spectra. These measurements also indicated that the disordered C-terminal regions (residues 98-140) of the membrane-bound states of both variants have similar structural and dynamical properties to those of αS WT. To gain further insights into the topological nature of mobile regions in the membrane-bound αS A30P and αS E46K variants, we used paramagnetic relaxation enhancement experiments28. By doping the SUVs with low levels (2%) of a lipid carrying an unpaired electron on the head group, namely the gadolinium salt of PE-DTPA (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid), we could observe selective line broadening of individual resonances in the INEPT spectrum of both variants ( Supplementary Fig. 5), revealing those residues that interact transiently with the membrane surface. The resulting broadening patterns of hydrophobic (including L100, A107, P108, I112, L113, P117, V118 and P120) and positively charged (K97 and K102) residues were found to be similar to those observed in αS WT. The selective broadening of the INEPT peaks of the C-terminal residues in these paramagnetic relaxation enhancement experiments is in agreement with the modest levels of saturation transfer that are detected for this region using CEST experiments performed with a bandwidth of 350 Hz and offset frequencies of ±1.5 kHz ( Supplementary Fig. 4a), which also provide evidence for transient tethering of the C-terminal region of αS onto the membrane surface.

Mechanism of vesicle assembly promoted by αS

The solution-state and solid-state NMR measurements described above reveal a striking degree of independence between the membrane-binding properties of the N-terminal membrane-anchor region of αS, which is significantly affected by the A30P and E46K mutations, and of the region spanning residues 65-97, which instead shows negligible differences as a result of these mutations ( Supplementary Figs 3 and 4). These independent membrane-binding modes suggest that, in addition to interacting with the membrane surface of the same SUV, these two regions are sufficiently independent to bind simultaneously two different SUVs. Indeed, our modelling studies show that a single αS molecule could bind and bridge two vesicles that are as much as 150 Å apart ( Fig. 3a), with both the N-terminal anchor region (the N-terminal 25 residues) and the central region of αS (residues 65-97) adopting the conformations of amphipathic α-helices. These data, therefore, provide the structural basis of the mechanism by which αS promotes the interaction between vesicles that has been observed experimentally both in vitro 14,15 and in vivo 13,16.

( a) Molecular details of the double-anchor mechanism described in this work. SUVs of 50 nm in diameter were modelled to mimic as closely as possible the experimental conditions in this study (see Methods section). αS was modelled with the N-terminal anchor in an amphipathic α-helical conformation (red) and bound to the lower vesicle. The region 65-97 (cyan) of αS was modelled in an amphipathic α-helical conformation bound to the upper vesicle. The C-terminal fragment (residues 98-140) and the linker region 26-59 are shown in pink and grey colours, respectively. With this topology the modelling reveals that a single αS molecule could simultaneously bind two vesicles that are up to 150 Å apart. ( b, c) Cryo-EM ( b) and STED ( c) images acquired on SUVs at a concentration of 0.5 mg ml −1. ( d, e) Cryo-EM ( d) and STED ( e) images measured on SUVs following a 12 h incubation with 200 μM αS WT. ( f, g) Cryo-EM ( f) and STED ( g) images acquired on SUVs following a 12 h incubation with 200 μM αS Sw.

To obtain further evidence of this 'double-anchor' mechanism ( Fig. 3a), we used our findings to design a further variant of αS having structural properties that we anticipated should enhance the probability of αS binding simultaneously to two different vesicles in such a way as to mediate their interaction. In particular, to favour the double-anchor mechanism ( Fig. 3a), this variant was designed to enhance the detachment of the region 65-97 from the membrane surface when αS is bound to the SUVs via its N-terminal anchor region. We identified for this purpose a swapped sequence (αS Sw) incorporating the E46K and K80E mutations ( Supplementary Fig. 6). In particular, by replacing the lysine at position 80 with a glutamic acid (K80E), the local binding to negatively charged vesicles is disfavoured, hence shifting the conformational equilibrium of the fragment 65-97 towards a state where this region is less strongly bound to the SUV surface. By contrast, because the K80E mutation also affects the overall membrane affinity of αS, a second mutation in which the glutamic acid at position 46 is replaced by a lysine (E46K) was selected to increase the interaction between the N-terminal anchor region and the SUV surface, as probed in αS E46K ( Figs 1 and 2), thereby restoring an overall K D comparable to that of the wild-type protein.

We tested experimentally whether or not αS Sw possessed the anticipated structural and thermodynamical properties characteristic of its membrane-bound state. In agreement with our design, we found the binding affinity of αS Sw for SUVs, measured by circular dichroism28, to be similar to that of αS WT ( Supplementary Fig. 7a-c). By contrast the structural properties of the αS Sw variant, as probed by CEST ( Supplementary Fig. 7d-f), showed a significant reduction in the membrane interaction of the central region (residues 65-97) of the variant than in αS WT. These data indicate that αS Sw binds SUVs with essentially the same overall affinity as αS WT but assumes different structural and dynamical properties in its bound state that promote an enhanced exposure of the segment 65-97. CEST also confirmed the stronger interaction of the anchor region of αS Sw compared with that of αS WT, which in the designed variant is extended to residue 42 as a consequence of the E46K mutation ( Supplementary Fig. 7e). As αS Sw and αS E46K have the same sequence except at position 80, we plotted the differences in the CEST profiles of these two variants; this comparison reveals clearly that the binding properties of these two variants to the SUVs are indistinguishable except in the region 65-97 ( Supplementary Fig. 8) thereby providing additional evidence for the independence of the membrane-binding properties of the N-terminal and central regions in αS.

Synaptic vesicle assembly induced by αSWT and αSSw

We compared the efficiency with which αS Sw and αS WT promote the interaction and assembly of vesicles by monitoring, using cryo-EM, the ability of the two variants to promote coalescence and fusion of synaptic-like SUVs in vitro 16. As a control, cryo-EM images of 0.05% DOPE:DOPS:DOPC SUVs incubated for 12 h in the absence of αS showed spherical vesicles of diameters ranging between 30 and 60 nm, with negligible evidence of vesicle fusion over the period of incubation ( Fig. 3b and Supplementary Fig. 9a), showing that in the absence of αS the spontaneous fusion of SUVs occurs with extremely slow kinetics. In the presence of αS, however, the SUVs experience a considerable number of fusion events, with cryo-EM images revealing the presence of elongated fused vesicles having long axes of up to 200 nm under the conditions used in this study ( Fig. 3d and Supplementary Fig. 9b). Incubating SUVs with αS Sw, however, resulted in a very significant increase in the extent of vesicle fusion, with cryo-EM images clearly indicating that this variant is significantly more active in promoting the interaction of SUVs ultimately leading to very large vesicle assemblies ( Fig. 3f and Supplementary Fig. 9c).

To generate a quantitative analysis of the sizes of the SUVs in the presence and absence of the αS variants, we used STED microscopy, which enabled us to sample rapidly the sizes of thousands of vesicles to achieve statistically significant differences in the distributions. STED imaging was carried out by fluorescently labelling the DOPE:DOPS:DOPC SUVs with 2% of DOPE labelled with the ATTO 647N fluorophore. Images of isolated vesicles resulted in a size distribution centred at 55±11 nm ( Fig. 3c and Supplementary Fig. 10a-b), within the range anticipated from the preparation protocol28. However, STED images of vesicles incubated with αS WT clearly revealed the presence of numerous clusters of fused lipids, whose sizes were quantified by using an algorithm that fits annular shapes to the lipid vesicles, rather than the 2D Gaussian fitting used in the case of isolated vesicles (Methods section and Supplementary Fig. 10). The resulting distributions indicate that SUVs incubated with αS WT give rise to two populations of vesicles, one with properties similar to those of vesicles imaged in the absence of αS and another attributable to fused vesicles, with a distribution centred at 115±30 nm and with a relative population of 16% of the imaged vesicles ( Fig. 3e and Supplementary Fig. 10c-d).

Following incubation with αS Sw, however, both the sizes and relative populations of the fused vesicles increased markedly, with the size distribution of fused vesicles now centred at 181±48 nm and with a relative population of 32% of the imaged vesicles ( Fig. 3g and Supplementary Fig. 10e-f). Similar conclusions to those obtained from STED analysis were obtained from measurements of dynamic light scattering (DLS), which showed that the average size of the SUVs increases in the presence of αS and that this effect is considerably greater with αS Sw than with αS WT ( Supplementary Fig. 11). Overall, the cryo-EM, STED and DLS data show that the engineered αS Sw variant has a very significantly enhanced activity in promoting the interactions between SUVs. As αS Sw and αS WT have the same amino acid composition and net charge, and bind SUVs with similar thermodynamic affinity, the enhanced interactions between vesicles on incubation with αS Sw can be attributed to the higher population of conformations with an enhanced exposure of the region 65-97 from the membrane surface, which increases the probability of associating with a different vesicle and to mediate the vesicle assembly via a double-anchor mechanism ( Fig. 3a), as probed by the fusion of DOPE:DOPS:DOPC SUVs on incubation with the protein ( Fig. 4).

The scheme shows the stepwise mechanism of vesicles assembly as probed from images obtained in vitro by cryo-EM, which are also shown. Disordered cytoplasmatic αS (red) binds dynamically to the surface of SUVs (green), as described in this study. SUVs coated with αS assemble with fast kinetics as a consequence of the double-anchor mechanism promoted by the αS molecules decorating their surfaces. The tethered SUVs, which are initially assembled together in dimeric, trimeric, tetrameric and higher order states, eventually fuse to form larger vesicles. With the increasing size of the fused vesicle, we observed preferential fusion events at the termini of the aggregated vesicles. This observation can be explained by the higher affinity of αS for significantly curved membrane surfaces19, which increases the concentration of bound αS at the termini of the elongated vesicles thereby promoting a stronger double-anchor mechanism in these loci.

To assess the role of the double-anchor mechanism in the clustering of synaptic vesicles (SVs) induced by αS, we incubated SVs purified from rat brain44,45 for 48 h at 37 °C in the presence or absence of αS samples (αS WT or αS Sw). The distribution of the sizes of the clusters of SVs on incubation were quantified using direct stochastic optical reconstruction microscopy (dSTORM)46 to acquire images on poly-L-lysine-coated glass plates. To visualize SVs, we used a primary antibody that is specific for the synaptic protein synaptotagmin 1 and therefore binds selectively to SVs, and a secondary antibody fluorescently labelled with ATTO 647N. The resulting dSTORM images ( Fig. 5a-c) were analysed by identifying the centres of mass of each of the SVs and then by applying a clustering approach47 to identify groups of SVs that, according to a distance cutoff of 60 nm, belong to the same cluster. The resulting distribution of sizes of SV clusters ( Fig. 5d) showed that 93% of the vesicles remain isolated after incubation for 48 h at 37 °C in the absence of αS, with the remaining 6% and 1% assembled in clusters consisting of two and three SVs, respectively. After incubation under the same conditions but in the presence of 85 μM of αS WT, up to 17% of the vesicles were clustered, some including assemblies composed up to five SVs (1%). In the presence of αS Sw, however, over 29% of the vesicles were observed to be clustered up to six SVs (2%). Cryo-EM images of the assembled structures ( Fig. 5d) revealed that the surfaces of the SVs composing the clusters are separated by distances that extend up to 15 nm, in agreement with the double-anchor mechanism ( Fig. 3a).

SVs purified from rat brain were incubated for 48 h at 37 °C. The concentrations during the incubation were 0.5 mg ml −1 and 85 μM for the SVs and the αS variants, respectively. ( a- c) dSTORM imaging of SVs alone ( a) and SVs incubated with αS WT ( b) and with αS Sw ( c). The images were collected using a previously described protocol46. Scale bars, 1 μm. To generate fluorescent SVs, we used a primary antibody that is specific for synaptotagmin 1 and a secondary antibody that is covalently linked to an ATTO 647 N dye. 10,000 fluorescence frames with an exposure time of 10 ms were recorded. The field of view imaged covered 1,997 × 1,997 camera pixels, corresponding to an area on the sample of ∼20 × 20 μm ². ( d) To assess the level of clustering of the SVs, we adapted an approach that has previously been successfully employed to analyse protein self-assembly.47 For each dSTORM image, clusters of SVs were identified on the basis of the distances between the centres of mass of the SVs. In particular two or more vesicles were associated with a specific cluster if their distances apart are less than 60 nm. The distribution of SVs in clusters of different sizes is reported using orange, green and blue histograms for SVs, SVs in the presence of αS WT and SVs in the presence of αS Sw, respectively. Cryo-EM images (scale bar, 50 nm) show representative clusters of different size.

It is widely recognized that the physiological activity of αS is associated with its ability to bind to a variety of lipid membranes48. A number of studies support an emerging view that a key biological role of αS is to mediate the interactions and assembly of synaptic vesicles14,16. Vesicle clustering by αS has been shown to be a vital step in many functional processes, including endoplasmic reticulum-to-Golgi vesicle trafficking13,49 and recycling of the SVs within the mechanism of neuronal communication11. In the present study, we have examined the structural principles underlying the αS-induced interactions and assembly of SVs by characterizing the membrane-binding properties of two mutational variants of αS linked with familial PD. This analysis reveals that two key regions of the protein, namely the N-terminal membrane-anchor (residues 1 to 25) and the central segment of the sequence (residues 65-97, having significant overlap with the NAC region), have independent membrane-binding properties and therefore are not only able to interact with a single SV, but can also simultaneously bind to two different vesicles ( Fig. 3a) thereby promoting their interaction and assembly as shown in vitro and in vivo 14,15,16. The resulting double-anchor mechanism explains why the deletion of the segment 71-82 in the NAC region of αS or the impairment of the membrane affinity of the N-terminal anchor region of the protein severely affects in vivo vesicle clustering as shown in Saccharomyces cerevisiae 16. This mechanism also provides a structural explanation for the suggested loss of function caused by A30P, which is associated with an impairment of vesicle clustering by αS A30P as observed both in vitro and in vivo 14,16, as well as for the functional regulation proposed to occur via the phosphorylation of serine 87 (ref. 50). In particular, by introducing a negative charge in the region 65-97, the phosphorylation of S87, which has been identified both in functional contexts and in the case of synucleinopathies, has similar effects to those of the K80E mutation in αS Sw.

We tested this molecular mechanism by engineering the mutational variant αS Sw, which was designed to enhance the probability of αS binding simultaneously to two different vesicles. Our studies of this variant have provided strong evidence in support of the proposed mechanism by showing that enhanced exposure of the central region, spanning residues 65-97 in the membrane-bound state of αS, promotes more strongly the clustering of SVs purified from rat brain ( Fig. 5) and the assembly and fusion of DOPE:DOPS:DOPC SUVs ( Fig. 4). It has previously been suggested that a broken α-helix structural topology of αS22,51, which is a conformation that αS adopts on binding to detergent micelles, could play a role in vesicle-vesicle interactions stimulated by αS48,52. The present study, however, shows experimentally that the underlying mechanism by which αS mediates the interactions between lipid vesicles relies on the balance between ordered (membrane-bound) and disordered (membrane-detached) conformational states of the region spanning residues 65-97 of the protein. Perturbing this balance, as we have done rationally with αS Sw, or on alteration of the expression levels of αS, can dramatically affect its ability to promote the vesicle assembly in vivo leading to defects in the regulation of vesicle trafficking11,13,49,53,54,55,56,57.

Other studies also suggest that αS could act as a molecular chaperone for the formation of SNARE complexes, which appears to result from the direct interaction between αS and synaptobrevin 2 at the surface of SVs14,58. Such an interaction was shown to be independent of the NAC region, suggesting that this region has no direct functional role in this particular process59. The present data, however, reveal that the NAC region is not only involved in αS aggregation, as extensive evidence has previously indicated4,60,61, but also has a specific role in a key molecular mechanism associated with the normal function of αS. This study provides evidence that the membrane affinity of the NAC region of αS is finely tuned to ensure an optimal degree of local detachment from the membrane surface to enable binding to occur between different vesicles. The present finding that, by perturbing this fine tuning through the design of the αS Sw variant, it is possible to promote stronger interactions between vesicles ( Figs 3 and 5) indicates that the exposure of the region 65-97 in the vesicle-bound state of αS is crucial for the physiological mechanism of SVs clustering and, at least in the case of αS Sw, has more relevance than the local membrane-binding affinity of this region, which in this variant is reduced as a result of the K80E mutation. The selection toward sequence properties of αS that enable the detachment of the amyloidogenic NAC region from the vesicle surface to favour the functional mechanism described in this study, however, can also lead to aberrant behaviour, as these conformational states are particularly vulnerable to self-association leading to αS aggregation at membrane surfaces11,62,63,64,65. Taken together, these findings provide therefore a new mechanistic link between functional and pathological roles of αS.

αS purification

αS WT was expressed and purified in Escerichia coli using a pT7-7 plasmid in which αS gene is under the control of the phage T7 RNA polymerase promoter. BL21 (DE3)-gold competent cells (Agilent Technologies, Santa Clara, USA) were transformed with this plasmid using heat-shock and subsequently grown in an isotope-enriched M9 minimal medium containing 1 g l −1 of ¹⁵N ammonium chloride, 2 g l −1 of ¹³C-glucose and 100 μg ml −1 ampicillin (Sigma-Aldrich, St Louis, USA) to produce uniformly ¹⁵N and/or ¹³C labelled αS samples. Cell growth was carried out at 37 °C under constant shaking at 250 rpm to an OD600 of 0.6. Subsequently the expression of the protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C for 4 h and cells were then harvested by centrifugation at 6,200 g (Beckman Coulter, Brea, USA). The cell pellets were resuspended in lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA and EDTA-free complete protease inhibitor cocktail tablets obtained from Roche, Basel, Switzerland) and lysed by sonication. The cell lysate was centrifuged at 22,000 g for 30 min to remove cell debris and the supernatant was then heated for 20 min at 70 °C and subsequently centrifuged at 22,000 g to precipitate the heat-sensitive proteins. Subsequently streptomycin sulfate was added to the supernatant to a final concentration of 10 mg ml −1 to stimulate DNA precipitation. The mixture was stirred for 15 min at 4 °C followed by centrifugation at 22,000 g. Then, ammonium sulfate was added to the supernatant to a concentration of 360 mg ml −1 to precipitate the protein. The solution was stirred for 30 min at 4 °C and centrifuged again at 22,000 g. The resulting pellet was resuspended in 25 mM Tris-HCl, pH 7.7 and dialyzed against the same buffer to remove salts. The dialyzed solutions were then loaded onto an anion exchange column (26/10 Q sepharose high performance, GE Healthcare, Little Chalfont, UK) and eluted with a 0 to 1 M NaCl step gradient. αS was eluted at ∼300 mM NaCl and then further purified by loading onto a size-exclusion column (Hiload 26/60 Superdex 75 preparation grade, GE Healthcare, Little Chalfont, UK). All the fractions containing the monomeric protein were pooled together and concentrated by using Vivaspin filter devices (Sartorius Stedim Biotech, Göttingen, Germany). The purity of the aliquots after each step was analysed by SDS-PAGE and the protein concentration was determined from the absorbance at 275 nm using an extinction coefficient of 5,600 M −1 cm −1.

To express and purify the mutational variants of αS (αS A30P, αS E46K and αS Sw), we employed the same experimental procedure as used in the case of αS WT. Single point mutations of the αS WT plasmid were obtained using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Inc., Ipswich, USA). Table 1 reports the primers employed to obtain the plasmids of αS A30P, αS E46K and αS Sw.

Preparation of synaptic-like SUVs

SUVs containing a molar ratio of 5:3:2 of DOPE:DOPS:DOPC (Avanti Polar Lipids, Inc., Alabaster, USA) were prepared from chloroform solutions of the lipids as described previously15,28. Briefly, the lipid mixture was evaporated under a stream of nitrogen gas and then dried thoroughly under vacuum to yield a thin lipid film. The dried thin film was re-hydrated by adding aqueous buffer (20 mM sodium phosphate, pH 6.0) at a concentration of 15 mg ml −1 (1.5%) and subjected to vortex mixing. In all NMR experiments described in this paper SUVs were obtained by using several cycles of freeze-thawing and sonication until the mixture became clear15,28. In the particular case of CEST experiments, SUVs at a concentration of 0.06% (0.6 mg ml −1) were mixed with αS samples after sonication. For ssNMR studies αS was added to the SUV mixtures up to a molar ratio of 1:65 protein:lipid. The mixtures were then pelleted at 300,000 g for 30 min at 4 °C (Beckman Coulter Optima TLX Inc. Brea, USA) by using a TLA 100.3 rotor. Subsequently the SUV-αS samples were transferred into 3.2 mm Zirconia XC thin-walled MAS rotors for ssNMR experiments. For STED and cryo-EM imaging experiments, as well as for DLS, DOPE:DOPS:DOPC SUVs were prepared by extrusion through membranes with a 50 nm pore diameter (Avanti Polar Lipids, Inc) after re-hydration in 20 mM sodium phosphate (pH 6.0) at a concentration of 1.0 mg ml −1 (0.1%).

Purification of SVs from rat brain

SVs composed of phospholipid molecules (amounting to 30% of their composition), proteins (58%) and cholesterol (12%)44,45 were purified from brains of rat provided by Charles River Laboratories (Animal work was approved by the Named Animal Care and Welfare Officer (NACWO) and the Ethics Review Committee of the University of Cambridge). Rat brains were homogenized in 4 mM HEPES and 320 mM sucrose buffer using protease inhibitors via ten strokes at 900 r.p.m. in a glass-Teflon homogenizer (Wheaton, Millville, USA)44,45. All steps were carried out at 4 °C or in ice. The homogenates were centrifuged at 1,000 g for 10 min and the supernatants were collected and further centrifuged at 15,000 g for 15 min. The supernatants from the second centrifugation (Sup-2) were stored at 4 °C. The pellets from the second centrifugation, which contained the synaptosomes, were lysed using ice-cold water by applying three strokes at 2,000 r.p.m. Subsequently, HEPES buffer solutions containing protease inhibitors were added to the lysates and the resulting solutions were centrifuged at 17,000  g for 15 min, and the supernatant was combined with the Sup-2 supernatants. The resulting mixtures were centrifuged at 48,000 g for 25 min and the supernatants were loaded onto a 0.7 M sucrose cushion and centrifuged at 133,000 g for 1 h. The bottom half of the sucrose cushion was pooled and centrifuged at 300,000 g for 2 h. The pellets were resuspended in buffer (100 mM Tris-HCl, pH 7.4, 100 mM KCl) and loaded onto a Sephacryl S-1000 size-exclusion chromatography column (100 × 1 cm) resulting in a distinctive peak of the SVs44,45. The SVs were then stained against specific SV antibodies, including synaptotagmin 1, synaptobrevin 2, by western blot44,45. To perform dSTORM analyses, the SVs were incubated with a primary antibody (dilution 1:1000) that specifically recognizes synaptotagmin 1 (105103, Synaptic Systems, Göttingen, Germany) and a secondary antibody (dilution 1:100) fluorescently labelled with Atto 647N (40839, Sigma-Aldrich, St Louis, USA).

CEST experiments

We employed CEST measurements28,36,37,38,40 to gain a deeper understanding of the equilibrium between membrane unbound and membrane-bound states of αS. In the study of αS-SUV interactions, CEST shows enhanced characteristics compared to standard heteronuclear correlation spectroscopy, including a significantly higher sensitivity at low lipid:protein ratios, conditions under which protein or lipid aggregation can be minimized64. The resulting NMR signals enable the interaction between αS and the membrane surface to be probed without interference from additional factors that may influence the transverse relaxation rates of the protein resonances36,37,38,40. In the CEST experiments employed here, a continuous weak radiofrequency field (either 350 or 170 Hz) was applied off-resonance (up to ±28 kHz) in the ¹⁵N channel, thereby saturating the broad spectroscopic transitions in the bound (undetectable) state but leaving the resonances of the free (detectable) state virtually unperturbed36,37,38,40. The saturation of the bound state was then transferred to the free state via chemical exchange, resulting in the attenuation of the intensities of the observable resonances in the visible unbound state. By carrying out a series of experiments at various offsets, it was possible to obtain a map of the strength of the interactions between the low (unbound αS) and high (SUV-bound αS) molecular weight species at a residue specific resolution.

Solution-state NMR experiments were carried out at 10 °C on Bruker spectrometers operating at ¹H frequencies of 700 MHz equipped with triple resonance HCN cryo-probes. CEST experiments were based on ¹H- ¹⁵N HSQC experiments by applying constant wave saturation in the ¹⁵N channel. Since we aimed at probing the exchange between monomeric αS (having sharp resonances) and αS bound to SUVs (having significantly broader resonances), a series of large offsets was employed (−28, −21, −14, −9, −5, −3, −1.5, 0, 1.5, 3, 5, 9, 14, 21 and 28 kHz), resulting in CEST profiles of symmetrical shapes ( Supplementary Fig. 1)28,36,37. An additional spectrum, saturated at −100 kHz, was recorded as a reference. The CEST experiments were recorded using a data matrix consisting of 2,048 (t ₂, ¹H) × 220 (t ₁, ¹⁵N) complex points. Assignments of the resonances in ¹H- ¹⁵N HSQC spectra of αS WT were derived from our previous studies28 whereas assignments of the mutational variants employed in this work were obtained by a standard combination of triple resonance solution NMR spectra66.

MAS measurements

MAS provides complementary information to CEST as it allows the protein resonances in the vesicle-bound state, which is inaccessible to solution-state NMR, to be probed directly. MAS experiments were carried out on a 16.85 T Bruker Spectrometer with a 3.2 mm E Free probe. DARR experiments42 were performed at a MAS rate of 10 kHz using a series of different mixing times (20, 50, 100, 200 and 500 ms), and the spectra were acquired at −19 and 4 °C (the latter for control experiments only) using a 1 ms contact time. INEPT spectra41 were measured at 4 °C using a MAS rate of 10 kHz. Pulse widths were 2.5 μs for ¹H and 5.5 μs for ¹³C, and ¹H TPPM decoupling was applied at ωRF/(2π)=71.4-100 kHz (ref. 28).

Cryo-EM measurements

All samples used in cryo-EM measurements were incubated, with or without αS (200 μM), for 12 h at 298 K using fresh preparations of DOPE:DOPS:DOPC SUVs at a concentration of 0.05%. After incubation cryo-EM grids were prepared by vitrifying the sample solutions using aliquots of 2 μl and a Vitrobot Mark IV (FEI, Hillsboro, USA) at a relative humidity of 100%. The samples were loaded on a glow-discharged Quantifoil Copper 300 mesh R2/2 grids (Quantifoil Micro Tools GmbH, Germany) and blotted with filter paper for 2.5 s to leave a thin film of solution. The blotted samples were plunged into liquid ethane and stored under liquid nitrogen before imaging. Samples were examined using a Philips CM200 FEG electron microscope operating at 200 kV (FEI, Hillsboro, USA), using a Gatan 626 cryo-holder (Gatan, Pleasantos, USA) cooled with liquid nitrogen to temperatures below −180 °C. Digital images were acquired on a TVIPS FC415 CCD camera using the EMMENU 4 software package (TVIPS, Munich, Germany).

STED microscopy

STED imaging67,68 was carried out by fluorescently labelling the DOPE:DOPS:DOPC SUVs with 2% of fluorescently labelled DOPE (ATTO 647N DOPE, ATTO-TECH, USA). STED microscopy allows the diffraction limit in optical microscopy (∼200 nm) to be overcome69 and imaging was performed on a home-built pulsed STED microscope69 using a single titanium-sapphire oscillator centred at λ STED=765 nm (Ti:S, Mai Tai HP, Spectra-physics, Santa Clara, USA) to generate the STED beam, which was subsequently split into two using a half-plate and a polarization beam splitter. Of these two beams, the one transmitted was focused onto a photonic crystal fibre (FemtoWhite, NKT Photonics, Cologne, Germany) to produce white light radiation. From this light, an excitation beam, centred at λ Exc=640 nm, was extracted using a bandpass filter (637/7 BrightLine HC, Semrock, NY, USA) and coupled into a 30 m long polarization maintaining single-mode fibre (PM630-HP, Thorlabs, Newton, UK). The reflected STED beam was passed through a 50 cm long glass block of SF66 and a 100 m long polarization maintaining single-mode fibre (PM-S630-HP, Thorlabs, Newton, UK) to stretch the pulse duration to ∼100-200 ps. In addition, the STED beam was converted into a so called donut beam by a spatial light modulator (X10468−02, Hamamatsu Photonics, Hamamatsu City, Japan). The excitation and STED beams were recombined with a dichroic mirror (T735spxr, Chroma, Bellow Falls, USA) and detected using a commercial point-scanning microscope (Abberior Instruments, Göttingen, Germany) comprising of a microscope frame (IX83, Olympus, Shinjiuku, Japan), a set of galvanometer mirrors (Quad scanner, Abberior Instruments, Göttingen,, Germany) and a detection unit. The beams were focused onto the sample by a × 100/1.4 NA oil immersion objective lens (UPLSAPO 100XO, Olympus, Göttingen, Germany) and images were acquired by raster scanning the beams across the sample using the Inspector software (Andreas Schönle, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). We used a field of view of 30 × 30 μm ² with a pixel size of 15 × 15 nm ² and a pixel dwell time of 20 μs. Fluorescence photons emerging from the sample were collected by the microscope objective lens, de-scanned by the galvanometer mirrors, focused onto a pinhole and detected using an avalanche photodiode (SPCM-AQRH, Excelitas Technologies. Waltham, USA). The laser powers, measured at the objective back aperture, were ca. 20 μW and 150 mW for the excitation beam and for the STED beam, respectively.

Analysis of STED images for vesicle size measurement

Vesicle sizes were estimated from STED images by using in-house Matlab scripts ( Supplementary data 1). First, images of isolated vesicles were identified and analysed using a fitting based on a 2D Gaussian function, by convolving the images with a Gaussian filter whose dimensions match the extension of the expected STED point spread function. The centres of the vesicles were identified by finding local maxima of the convolved images, excluding the local maxima corresponding to fused vesicles by means of a threshold applied on the peak intensities. A different fitting procedure was optimized in the case of assembled vesicles that appear as hollow shapes in the STED images, as for a vesicle larger than the lateral resolution of the STED microscope the number of dye molecules probed increases on the edge of the shell. To estimate the size of the clusters and their relative number compared to the non-fused vesicles, all the vesicles appearing as fused were fitted by annular functions having a Gaussian radial profile (amplitude, position, radius and offset) using a nonlinear least squares approach.

Direct stochastic optical reconstruction microscopy

Super-resolution imaging was performed using dSTORM microscopy with a Nikon Eclipse TE 300 inverted wide-field microscope using a × 100, 1.49-N.A total internal reflection fluorescence46 objective lens (Nikon Ltd., Kingston upon Thames, UK). The vesicle and αS samples were adhered to a glass coverslip coated in poly-L-lysine (P4707, Sigma-Aldrich, St Louis, USA) before photoswitching buffer solution was added, consisting of 100 mM mercaptoethylamine (MEA) in phosphate buffered saline (PBS, pH 8.2). For imaging, a laser emitting at a wavelength of 640 nm was used (Toptica Photonics AG, Graefelfing, Germany) for excitation of the Atto 647N dye. A 405 nm laser (Mitsubishi S3 Electronics Corp., Tokyo, Japan) was used as the reactivation source, which was only turned on when the number of active fluorophores in the field of view was visibly reduced. Imaging was performed under total internal reflection fluorescence illumination conditions, ensuring that the exact centre of the field of view was illuminated. The field of view covered 1,997 × 1,997 camera pixels, corresponding to an area on the sample of ∼20 × 20 μm ². 10,000 fluorescence frames were recorded, each corresponding to an exposure time of 10 ms; the latter was matched to be in the range of the average 'on' time of the fluorescent dyes. The fluorescence light in the detection path was filtered and imaged with an Ixon DV887 ECS-BV EM-CCD camera (Andor, Belfast, UK). The image analysis was performed using frames 1000 to 10 000 in each sequence. From each image stack, a reconstructed dSTORM image was generated using the open-source rapidSTORM software ( Supplementary Data 2) developed in house using MATLAB (The MathWorks, Inc.).

Dynamic light scatterning

DLS measurements of vesicle size distributions were performed using a Zetasizer Nano ZSP instrument (Malvern Instruments, Malvern, UK) with backscatter detection at a scattering angle of 173°. The viscosity (0.8882 cP) and the refractive index (1.330) of water were used as parameters for the buffer solution, and the material properties of the analyte were set to those of the lipids (absorption coefficient of 0.001 and refractive index of 1.440). SUVs were used at a concentration of 0.05% in these measurements and the experiments were performed at 25 °C. The acquisition time for the collection of each dataset was 10 s and accumulation of the correlation curves was obtained using 10 repetitions. Each measurement was repeated 10 times to estimate standard deviations and average values of the centres of the size distributions ( Supplementary Fig. 11).

Modelling

Schematic representations of αS bound to SUVs were obtained by using molecular dynamics (MD) simulations in implicit solvent. The structure of αS in the double-anchor mechanism ( Fig. 3a) were obtained by starting from the model of membrane-bound conformation αS characterized by an elongated helix (residues 1 to 97) with a disordered C-terminal region (residues 98-140) which was part of the ensemble characterized previously28. Atomic coordinates (in Cartesian space) of the N-terminal anchor were harmonically restrained to maintain a fixed position whereas the region spanning residues 65-97 was restrained in the alpha-helical conformation. A constant force along the membrane normal was applied to this region to extend it toward the second vesicle (up in the Fig. 3a). The reminder of the protein (residues 26-59 and 98-140) was allowed to relax under the Newtonian laws of motions during the MD simulations. Curved vesicle surfaces were generated by starting from atomic models of DOPE:DOPS:DOPC bilayers and by generating roto-translations that imposed a spherical symmetry with a radius of 25 nm.

Data availability

Data supporting the findings of this study are available within the article and its Supplementary Information Files and from the corresponding author on request.

How to cite this article: Fusco, G. et al. Structural basis of synaptic vesicle assembly promoted by α-synuclein. Nat. Commun. 7:12563 doi: 10.1038/ncomms12563 (2016).

This research was supported by Parkinson's UK (H-1103 to G.F. and C.M.D.), The Wellcome Trust (104933/Z/14/Z to A.D. and 089703/Z/09/Z to C.F.K.), the UK Medical Research Council (MR/N000676/1 to G.F., M.V., C.M.D., A.D. and MR/K015850/1 and MR/K02292X/1 to C.F.K.) and the UK Engineering and Physical Sciences Research Council (EP/H018301/1 to C.F.K.).

Author notes

Pierre Mahou

Present address: Present address: Laboratory for Optics and Biosciences, Ecole Polytechnique, Palaiseau, 91128 Cedex, France,

Authors and Affiliations

1. Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK

Giuliana Fusco, Ana Rita Costa, Michele Vendruscolo & Christopher M. Dobson

2. Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK

Tillmann Pape & Alfonso De Simone

3. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK

Amberley D. Stephens, Pierre Mahou, Clemens F. Kaminski & Gabriele S. Kaminski Schierle

4. Department of Chemistry & Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, 55455, Minnesota, USA

Gianluigi Veglia

Contributions

G.F., C.M.D. and A.D. designed the research. G.F., G.S.K., G.V., C.M.D. and A.D. designed the experiments. G.F., T.P., A.D.S., P.M., A.C. and A.D. performed the experiments. G.F., T.P., A.D.S., P.M., C.F.K., G.S.K., M.V., G.V., C.M.D. and A.D. analysed the data. G.F., C.M.D. and A.D., wrote the manuscript. All authors revised the manuscript critically and approved the final version.

Corresponding authors

Correspondence to Christopher M. Dobson or Alfonso De Simone.

Competing interests

The authors declare no competing financial interests.

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
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# data[].attributes.attachment_link.title
Structural basis of synaptic vesicle assembly promoted by α-synuclein - Nature Communications

# data[].attributes.attachment_link.description
α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly.

# data[].attributes.body
Some residues are stably bound (low I/I₀)

Other residues are in exchange between bound and unbound (intermediate I/I₀)

# data[].attributes.body
Can control the strength and 'location' of the B1 field, monitor the intensity of a particular peak

B1 field on top of peak --> sharp drop in intensity, recovers when B1 field moves off peak (intensity vs offset plot)

Large MW --> faster relaxation --> broader peak

If exchange occurs, the drop in intensity occurs before you actually hit the main peak and takes longer to recover (broad drop rather than fast drop)

Allows you to indirectly observe the broad intensity by monitoring the change in intensity of the main peak

# data[].attributes.headline
N terminus of αS: Exchange with large species 

# data[].attributes.subject
N terminus of αS: Exchange with large species 

# data[].attributes.body
Broad peak for N terminus --> indicates exchange with high MW species (binding the membrane)

# data[].attributes.headline
C terminus of αS: No exchange with large species 

# data[].attributes.subject
C terminus of αS: No exchange with large species 

# data[].attributes.body
Sharp peak for C terminus (saturation only around 0 offset) --> indicates no exchange (remains disordered, doesn't bind membrane)

# data[].attributes.headline
Mechanisms of amyloid formation revealed by solution NMR

# data[].attributes.subject
Mechanisms of amyloid formation revealed by solution NMR

# data[].attributes.headline
Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR

# data[].attributes.subject
Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR

# data[].attributes.body
CEST is an NMR method to probe exchange of NMR visible species with excited states

If the excited state has a very large MW, the method is also called dark exchange saturation transfer (DEST)

Based on the application of a weak saturating field (B1) at various places (offsets) on the spectrum

When an NMR resonance is saturated (i.e. when the B1 field is applied at its frequency, offset ~0), its intensity is diminished (dark blue)

If there is exchange with another conformation that has a large MW, saturation of the main observable species is observed at large offsets (light blue)

This happens because saturation of the large excited state is transferred to the small NMR observable main state through chemical exchange

# data[].attributes.body
NMR offers many opportunities to study weak biomolecular interactions as NMR observables are sensitive to weak binding

# data[].attributes.body
Also membrane contacts for the C terminal disordered region

# data[].attributes.headline
Topological properties of αS relative to the membrane with PRE

# data[].attributes.subject
Topological properties of αS relative to the membrane with PRE

# data[].attributes.body
Attach spin labels to membrane --> changes (increased R2) observed in peaks corresponding to residues involved in binding the membrane

N terminal anchor: Membrane contacts in the rigid region of the protein

# data[].attributes.headline
Topological properties of proteins relative to the membrane 

# data[].attributes.subject
Topological properties of proteins relative to the membrane 

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αS bound to SUVs has three regions with different structural and dynamical regimes

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αS bound to SUVs has three regions with different structural and dynamical regimes

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N-terminal 25 residues --> rigid helix

Residues 26-97 --> intermediate behaviour (dynamics/structure)

C terminal residues 98-140 --> unstructured and highly dynamical

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Structural basis of synaptic vesicle assembly promoted by α-synuclein

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Structural basis of synaptic vesicle assembly promoted by α-synuclein

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α-Synuclein (αS) is a 140-residue protein whose aggregation has been strongly associated with Parkinson's disease (PD)1,2,3,4,5. Fibrillar deposits of αS are the major constituents of Lewy bodies6,7,8, a hallmark of the disease, and inherited forms of early onset PD are associated with mutations, duplications and triplications of the αS-encoding gene9,10. Despite the general consensus on its pathological relevance, the physiological role of αS remains widely debated. In this context, a view is emerging in which αS is involved in the dynamics of synaptic vesicle (SV) trafficking by regulating a distal reserve pool of SVs that controls the amount of vesicles docked at the synapses during neurotransmitter release11,12. This biological role is directly associated with the ability of αS to bind to synaptic vesicles and induce their interaction and assembly in vitro and in vivo 13,14,15,16. Indeed, in dopaminergic neurons αS exists in a tightly regulated equilibrium17 between a cytosolic monomeric form, which is predominantly disordered18,19,20, and a membrane-bound state, which is rich in α-helix structure in the region spanning residue 1-90 of the protein sequence15,21,22,23,24,25,26.

Because of its intrinsic ability to bind to a wide variety of biological membranes, the physiological state of membrane-bound αS is extremely difficult to characterize, as a variety of factors, including the presence of detergents22 and chemical modification of the protein27, can alter dramatically the structural properties of its bound state15. In a recent study, three major regions were identified to have distinct structural and dynamical properties that influence in different ways the nature of the membrane-bound state of αS28; these regions include an N-terminal α-helical segment, acting as the membrane-anchor, an unstructured C-terminal region, weakly associated with the membrane, and a central region, undergoing order-disorder transitions in the membrane-bound state and determining the affinity of αS for lipid bilayers of different composition28. This structural variability indicates that it is of fundamental importance to investigate the binding of αS to lipid membranes under conditions that reproduce as closely as possible the physiological environment relevant to that of presynaptic vesicles15.

We describe here a detailed characterization of the dynamical and structural properties at the surface of synaptic-like lipid vesicles of two familial αS mutations that have opposite effects on its affinity for membrane binding29,30. On the basis of these studies, we characterized the details of the underlying mechanism by which a single molecule of αS binds two different synaptic vesicles and promotes their interaction and assembly. This mechanism, which involves a double-anchoring step enabling αS to form a dynamic link between two vesicles, is strongly supported by an experiment in which a variant of αS was engineered to adopt structural properties in its membrane-bound state that result in enhanced αS-mediated interactions between synaptic vesicles while maintaining the same amino acid composition, charge and membrane-binding affinity of the wild-type protein. The mechanism, which was verified using both synthetic lipid vesicles and synaptic vesicles purified from rat brain, provides evidence that the specific level of affinity for membrane binding of the non-amyloid-β component (NAC) region of αS is a fundamental functional property enabling this protein to mediate the interaction between vesicles.

Binding of αS variants to membranes

Using solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with cryo-electron microscopy (cryo-EM) and stimulated emission depletion (STED) imaging, we have characterized the structural properties at the surface of synaptic-like vesicles of the familial αS mutants A30P31 (αS A30P) and E46K32 (αS E46K) and compared their behaviour with that of the wild-type protein (αS WT)28. In particular, we studied the interactions of αS A30P, αS E46K and αS WT with small unilamellar vesicles (SUVs) composed of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in 5:3:2 molar ratios29, as such SUVs have been reported to be good mimics of synaptic vesicles for composition and curvature15. The combination of magic angle spinning (MAS33,34) measurements in solid-state NMR (ssNMR35) and chemical exchange saturation transfer (CEST28,36,37,38,39,40, Supplementary Fig. 1) experiments in solution-state NMR used in this study has already been shown to be highly effective in probing interactions between αS and SUVs, enabling the degree of order and disorder in the membrane-bound state of αS WT to be characterized28.

In the analysis of the interaction between αS A30P and SUVs, CEST profiles ( Fig. 1 and Supplementary Fig. 2) provided detailed information concerning the effects of the A30P mutation, which was shown to reduce very substantially the binding affinity of αS for SUVs15. In αS A30P, indeed, the binding of the N-terminal anchor region was found to involve a smaller number of residues than in the case of αS WT (the N-terminal 20 residues compared with the N-terminal 25 residues in αS WT) with generally a lower degree of CEST saturation than in the case of αS WT. The major differences between αS A30P and αS WT were evident in this membrane-anchor N-terminal region, while the remainder of the protein sequence showed very limited variations in the CEST profiles of these two proteins. The opposite behaviour was observed for the αS E46K variant, which binds SUVs with higher affinity than does αS WT 15. Indeed, the CEST data indicate a significantly stronger interaction with the membrane for the N-terminal anchor region of αS E46K, which in this case extends up to residue 42 with a generally higher degree of saturation than in the case of αS WT. As with αS A30P, only marginal variations in the CEST profiles were observed in other regions of the sequence of αS E46K. The differences in the CEST saturation profiles of the two mutants compared with αS WT ( Supplementary Figs 3 and 4) indicate more specifically that the major changes in the modes of binding to SUVs of these mutational variants are associated primarily with the N-terminal region of the protein.

CEST experiments were recorded at a ¹H frequency of 700 MHz (see Methods section), using protein concentrations of 300 μM and 0.06% (0.6 mg ml −1) of DOPE:DOPS:DOPC lipids in a ratio of 5:3:2 and assembled into SUVs. ¹H- ¹⁵N HSQC spectra were recorded by using continuous wave saturation (170 Hz or 350 Hz) in the ¹⁵N channel at offsets ranging between −28 kHz and +28 kHz; an additional spectrum, saturated at −100 kHz, was recorded as a reference. Data recorded using a saturation bandwidth of 350 Hz are shown here (the data measured using a saturation bandwidth of 170 Hz are shown in Supplementary Fig. 2). For comparison, the plots in panels b and e are drawn using αS WT data from our previous investigation28. ( a- c) CEST surfaces for αS A30P ( a) αS WT 28 ( b) and αS E46K ( c). ( d- f) CEST saturation along the sequences of αS A30P ( d), αS WT 28 ( e) and αS E46K ( f). The green lines refer to the averaged CEST profiles measured using offsets at +/− 1.5 kHz, and the profiles for +/− 3 kHz and +/− 5 kHz are shown in black and red, respectively. ( g- i) Schematic illustration (see Materials and Methods) of the equilibrium between surface attached/detached local conformations in the membrane-bound states αS A30P ( g) αS WT 28 ( h) and αS E46K ( i). The major differences in the data of αS A30P, αS WT and αS E46K are located in the anchor region. Overall, these three variants of αS maintain the same topological properties at the surfaces of synaptic-like SUVs.

Topology of αSA30P and αSE46K bound to SUVs

To obtain detailed information on the topology of αS A30P and αS E46K when bound to the surface of SUVs, we used MAS ssNMR experiments. ¹³C- ¹⁵N-labelled αS samples were mixed with SUVs, as described previously,15,28 to reach a protein:lipid ratio of 1:65 (ref. 28). Under these conditions we could observe directly the resonances of both rigid and dynamical regions of the membrane-bound αS molecule by using cross polarization and insensitive nuclei enhanced by polarization transfer (INEPT) experiments41, respectively. In the cross polarization regime, we performed ¹³C- ¹³C dipolar-assisted rotational resonance (DARR)42 measurements to detect homonuclear correlations between carbon atoms of residues strongly anchored to the membrane ( Fig. 2a). In our previous study of αS WT 28, the ¹³C- ¹³C DARR spectra identified resonances of residues 6-25 of the anchor region, showing that this region folds into a highly rigid α-helix lying essentially parallel to the membrane surface28. The ¹³C- ¹³C DARR spectra of the membrane-bound states of αS A30P and αS E46K are, however, substantially different from those of αS WT, indicating that the dynamical and structural properties of the anchor region vary considerably between the wild-type and variant forms of αS.

( a) ¹³C- ¹³C DARR correlation spectra (aliphatic regions) recorded at −19 °C using a 50 ms mixing time at a MAS rate of 10 kHz. We used a 1:65 protein:lipid ratio in both cases, and spectra of αS A30P and αS E46K are shown in the left and right panels, respectively. Residues are indicated using the single letter convention. The highest signal intensities in the spectra of the samples studied here were obtained by performing the measurements at −19 °C. Under these conditions the lipid mixtures used here are in the gel phase70, enabling ¹³C- ¹³C DARR spectra to be measured with significantly increased signal-to-noise ratios but without affecting the pattern of chemical shifts; the latter are consistent with those measured at 4 °C (ref. 28). No variations in the number of observed resonances or in the chemical shifts were observed using protein:lipid ratios ranging from 1:30 to 1:200 (ref. 28). ( b) ¹H- ¹³C correlation via INEPT transfer recorded at 4 °C at a MAS rate of 10 kHz. The experiments were performed at a ¹H frequency of 700 MHz using a 3.2 mm E Free probe. Atom names ca, cb, cg, cd and ce are used for C α, C β, C γ, C δ and C ɛ atoms, respectively.

In the case of αS A30P, the ¹³C- ¹³C DARR spectrum showed a very limited signal-to-noise ratio and almost a complete absence of cross peaks, suggesting that the anchor region of this variant is significantly more dynamic than the same region of the wild-type protein. In contrast, the ¹³C- ¹³C DARR spectrum of αS E46K showed a higher signal-to-noise ratio and a significantly larger number of intense cross peaks, indicating an elongated anchor region in this mutational variant that binds more strongly to SUVs. Using the dipolar connectivities from ¹⁵N- ¹³C cross polarization-based experiments, along with our previous assignment of αS WT and ¹³C- ¹³C DARR spectra acquired at different mixing times, we were able to assign individual spin systems in the ¹³C- ¹³C DARR spectra of αS A30P and αS E46K ( Fig. 2a). The chemical shifts were then compared with those obtained from solution-state NMR studies of αS WT in SDS and SLAS micelles22,43, and indicate that, despite the differences in dynamics and in the binding strength relative to lipid membranes, all the variants analysed here adopt a helical conformation at the N-terminal anchor when bound to membranes. This finding is particularly relevant in the case of the A30P mutation as it shows that, like the other variants and despite the insertion of a helix-breaker residue, αS A30P binds the lipid bilayer by adopting an amphipathic α-helix conformation at the N terminus and not as a disordered state that is tethered onto the lipid surface.

The highly dynamical regions of αS A30P and αS E46K bound to SUVs were then probed directly by INEPT measurements acquired using MAS ssNMR experiments41 and revealed ¹H- ¹³C correlations for resonances of the disordered C-terminal region of αS ( Fig. 2b), which is only weakly associated with the membrane28. In contrast to the results obtained from the DARR spectra, no significant differences were found between αS A30P and αS E46K in the INEPT spectra. These measurements also indicated that the disordered C-terminal regions (residues 98-140) of the membrane-bound states of both variants have similar structural and dynamical properties to those of αS WT. To gain further insights into the topological nature of mobile regions in the membrane-bound αS A30P and αS E46K variants, we used paramagnetic relaxation enhancement experiments28. By doping the SUVs with low levels (2%) of a lipid carrying an unpaired electron on the head group, namely the gadolinium salt of PE-DTPA (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid), we could observe selective line broadening of individual resonances in the INEPT spectrum of both variants ( Supplementary Fig. 5), revealing those residues that interact transiently with the membrane surface. The resulting broadening patterns of hydrophobic (including L100, A107, P108, I112, L113, P117, V118 and P120) and positively charged (K97 and K102) residues were found to be similar to those observed in αS WT. The selective broadening of the INEPT peaks of the C-terminal residues in these paramagnetic relaxation enhancement experiments is in agreement with the modest levels of saturation transfer that are detected for this region using CEST experiments performed with a bandwidth of 350 Hz and offset frequencies of ±1.5 kHz ( Supplementary Fig. 4a), which also provide evidence for transient tethering of the C-terminal region of αS onto the membrane surface.

Mechanism of vesicle assembly promoted by αS

The solution-state and solid-state NMR measurements described above reveal a striking degree of independence between the membrane-binding properties of the N-terminal membrane-anchor region of αS, which is significantly affected by the A30P and E46K mutations, and of the region spanning residues 65-97, which instead shows negligible differences as a result of these mutations ( Supplementary Figs 3 and 4). These independent membrane-binding modes suggest that, in addition to interacting with the membrane surface of the same SUV, these two regions are sufficiently independent to bind simultaneously two different SUVs. Indeed, our modelling studies show that a single αS molecule could bind and bridge two vesicles that are as much as 150 Å apart ( Fig. 3a), with both the N-terminal anchor region (the N-terminal 25 residues) and the central region of αS (residues 65-97) adopting the conformations of amphipathic α-helices. These data, therefore, provide the structural basis of the mechanism by which αS promotes the interaction between vesicles that has been observed experimentally both in vitro 14,15 and in vivo 13,16.

( a) Molecular details of the double-anchor mechanism described in this work. SUVs of 50 nm in diameter were modelled to mimic as closely as possible the experimental conditions in this study (see Methods section). αS was modelled with the N-terminal anchor in an amphipathic α-helical conformation (red) and bound to the lower vesicle. The region 65-97 (cyan) of αS was modelled in an amphipathic α-helical conformation bound to the upper vesicle. The C-terminal fragment (residues 98-140) and the linker region 26-59 are shown in pink and grey colours, respectively. With this topology the modelling reveals that a single αS molecule could simultaneously bind two vesicles that are up to 150 Å apart. ( b, c) Cryo-EM ( b) and STED ( c) images acquired on SUVs at a concentration of 0.5 mg ml −1. ( d, e) Cryo-EM ( d) and STED ( e) images measured on SUVs following a 12 h incubation with 200 μM αS WT. ( f, g) Cryo-EM ( f) and STED ( g) images acquired on SUVs following a 12 h incubation with 200 μM αS Sw.

To obtain further evidence of this 'double-anchor' mechanism ( Fig. 3a), we used our findings to design a further variant of αS having structural properties that we anticipated should enhance the probability of αS binding simultaneously to two different vesicles in such a way as to mediate their interaction. In particular, to favour the double-anchor mechanism ( Fig. 3a), this variant was designed to enhance the detachment of the region 65-97 from the membrane surface when αS is bound to the SUVs via its N-terminal anchor region. We identified for this purpose a swapped sequence (αS Sw) incorporating the E46K and K80E mutations ( Supplementary Fig. 6). In particular, by replacing the lysine at position 80 with a glutamic acid (K80E), the local binding to negatively charged vesicles is disfavoured, hence shifting the conformational equilibrium of the fragment 65-97 towards a state where this region is less strongly bound to the SUV surface. By contrast, because the K80E mutation also affects the overall membrane affinity of αS, a second mutation in which the glutamic acid at position 46 is replaced by a lysine (E46K) was selected to increase the interaction between the N-terminal anchor region and the SUV surface, as probed in αS E46K ( Figs 1 and 2), thereby restoring an overall K D comparable to that of the wild-type protein.

We tested experimentally whether or not αS Sw possessed the anticipated structural and thermodynamical properties characteristic of its membrane-bound state. In agreement with our design, we found the binding affinity of αS Sw for SUVs, measured by circular dichroism28, to be similar to that of αS WT ( Supplementary Fig. 7a-c). By contrast the structural properties of the αS Sw variant, as probed by CEST ( Supplementary Fig. 7d-f), showed a significant reduction in the membrane interaction of the central region (residues 65-97) of the variant than in αS WT. These data indicate that αS Sw binds SUVs with essentially the same overall affinity as αS WT but assumes different structural and dynamical properties in its bound state that promote an enhanced exposure of the segment 65-97. CEST also confirmed the stronger interaction of the anchor region of αS Sw compared with that of αS WT, which in the designed variant is extended to residue 42 as a consequence of the E46K mutation ( Supplementary Fig. 7e). As αS Sw and αS E46K have the same sequence except at position 80, we plotted the differences in the CEST profiles of these two variants; this comparison reveals clearly that the binding properties of these two variants to the SUVs are indistinguishable except in the region 65-97 ( Supplementary Fig. 8) thereby providing additional evidence for the independence of the membrane-binding properties of the N-terminal and central regions in αS.

Synaptic vesicle assembly induced by αSWT and αSSw

We compared the efficiency with which αS Sw and αS WT promote the interaction and assembly of vesicles by monitoring, using cryo-EM, the ability of the two variants to promote coalescence and fusion of synaptic-like SUVs in vitro 16. As a control, cryo-EM images of 0.05% DOPE:DOPS:DOPC SUVs incubated for 12 h in the absence of αS showed spherical vesicles of diameters ranging between 30 and 60 nm, with negligible evidence of vesicle fusion over the period of incubation ( Fig. 3b and Supplementary Fig. 9a), showing that in the absence of αS the spontaneous fusion of SUVs occurs with extremely slow kinetics. In the presence of αS, however, the SUVs experience a considerable number of fusion events, with cryo-EM images revealing the presence of elongated fused vesicles having long axes of up to 200 nm under the conditions used in this study ( Fig. 3d and Supplementary Fig. 9b). Incubating SUVs with αS Sw, however, resulted in a very significant increase in the extent of vesicle fusion, with cryo-EM images clearly indicating that this variant is significantly more active in promoting the interaction of SUVs ultimately leading to very large vesicle assemblies ( Fig. 3f and Supplementary Fig. 9c).

To generate a quantitative analysis of the sizes of the SUVs in the presence and absence of the αS variants, we used STED microscopy, which enabled us to sample rapidly the sizes of thousands of vesicles to achieve statistically significant differences in the distributions. STED imaging was carried out by fluorescently labelling the DOPE:DOPS:DOPC SUVs with 2% of DOPE labelled with the ATTO 647N fluorophore. Images of isolated vesicles resulted in a size distribution centred at 55±11 nm ( Fig. 3c and Supplementary Fig. 10a-b), within the range anticipated from the preparation protocol28. However, STED images of vesicles incubated with αS WT clearly revealed the presence of numerous clusters of fused lipids, whose sizes were quantified by using an algorithm that fits annular shapes to the lipid vesicles, rather than the 2D Gaussian fitting used in the case of isolated vesicles (Methods section and Supplementary Fig. 10). The resulting distributions indicate that SUVs incubated with αS WT give rise to two populations of vesicles, one with properties similar to those of vesicles imaged in the absence of αS and another attributable to fused vesicles, with a distribution centred at 115±30 nm and with a relative population of 16% of the imaged vesicles ( Fig. 3e and Supplementary Fig. 10c-d).

Following incubation with αS Sw, however, both the sizes and relative populations of the fused vesicles increased markedly, with the size distribution of fused vesicles now centred at 181±48 nm and with a relative population of 32% of the imaged vesicles ( Fig. 3g and Supplementary Fig. 10e-f). Similar conclusions to those obtained from STED analysis were obtained from measurements of dynamic light scattering (DLS), which showed that the average size of the SUVs increases in the presence of αS and that this effect is considerably greater with αS Sw than with αS WT ( Supplementary Fig. 11). Overall, the cryo-EM, STED and DLS data show that the engineered αS Sw variant has a very significantly enhanced activity in promoting the interactions between SUVs. As αS Sw and αS WT have the same amino acid composition and net charge, and bind SUVs with similar thermodynamic affinity, the enhanced interactions between vesicles on incubation with αS Sw can be attributed to the higher population of conformations with an enhanced exposure of the region 65-97 from the membrane surface, which increases the probability of associating with a different vesicle and to mediate the vesicle assembly via a double-anchor mechanism ( Fig. 3a), as probed by the fusion of DOPE:DOPS:DOPC SUVs on incubation with the protein ( Fig. 4).

The scheme shows the stepwise mechanism of vesicles assembly as probed from images obtained in vitro by cryo-EM, which are also shown. Disordered cytoplasmatic αS (red) binds dynamically to the surface of SUVs (green), as described in this study. SUVs coated with αS assemble with fast kinetics as a consequence of the double-anchor mechanism promoted by the αS molecules decorating their surfaces. The tethered SUVs, which are initially assembled together in dimeric, trimeric, tetrameric and higher order states, eventually fuse to form larger vesicles. With the increasing size of the fused vesicle, we observed preferential fusion events at the termini of the aggregated vesicles. This observation can be explained by the higher affinity of αS for significantly curved membrane surfaces19, which increases the concentration of bound αS at the termini of the elongated vesicles thereby promoting a stronger double-anchor mechanism in these loci.

To assess the role of the double-anchor mechanism in the clustering of synaptic vesicles (SVs) induced by αS, we incubated SVs purified from rat brain44,45 for 48 h at 37 °C in the presence or absence of αS samples (αS WT or αS Sw). The distribution of the sizes of the clusters of SVs on incubation were quantified using direct stochastic optical reconstruction microscopy (dSTORM)46 to acquire images on poly-L-lysine-coated glass plates. To visualize SVs, we used a primary antibody that is specific for the synaptic protein synaptotagmin 1 and therefore binds selectively to SVs, and a secondary antibody fluorescently labelled with ATTO 647N. The resulting dSTORM images ( Fig. 5a-c) were analysed by identifying the centres of mass of each of the SVs and then by applying a clustering approach47 to identify groups of SVs that, according to a distance cutoff of 60 nm, belong to the same cluster. The resulting distribution of sizes of SV clusters ( Fig. 5d) showed that 93% of the vesicles remain isolated after incubation for 48 h at 37 °C in the absence of αS, with the remaining 6% and 1% assembled in clusters consisting of two and three SVs, respectively. After incubation under the same conditions but in the presence of 85 μM of αS WT, up to 17% of the vesicles were clustered, some including assemblies composed up to five SVs (1%). In the presence of αS Sw, however, over 29% of the vesicles were observed to be clustered up to six SVs (2%). Cryo-EM images of the assembled structures ( Fig. 5d) revealed that the surfaces of the SVs composing the clusters are separated by distances that extend up to 15 nm, in agreement with the double-anchor mechanism ( Fig. 3a).

SVs purified from rat brain were incubated for 48 h at 37 °C. The concentrations during the incubation were 0.5 mg ml −1 and 85 μM for the SVs and the αS variants, respectively. ( a- c) dSTORM imaging of SVs alone ( a) and SVs incubated with αS WT ( b) and with αS Sw ( c). The images were collected using a previously described protocol46. Scale bars, 1 μm. To generate fluorescent SVs, we used a primary antibody that is specific for synaptotagmin 1 and a secondary antibody that is covalently linked to an ATTO 647 N dye. 10,000 fluorescence frames with an exposure time of 10 ms were recorded. The field of view imaged covered 1,997 × 1,997 camera pixels, corresponding to an area on the sample of ∼20 × 20 μm ². ( d) To assess the level of clustering of the SVs, we adapted an approach that has previously been successfully employed to analyse protein self-assembly.47 For each dSTORM image, clusters of SVs were identified on the basis of the distances between the centres of mass of the SVs. In particular two or more vesicles were associated with a specific cluster if their distances apart are less than 60 nm. The distribution of SVs in clusters of different sizes is reported using orange, green and blue histograms for SVs, SVs in the presence of αS WT and SVs in the presence of αS Sw, respectively. Cryo-EM images (scale bar, 50 nm) show representative clusters of different size.

It is widely recognized that the physiological activity of αS is associated with its ability to bind to a variety of lipid membranes48. A number of studies support an emerging view that a key biological role of αS is to mediate the interactions and assembly of synaptic vesicles14,16. Vesicle clustering by αS has been shown to be a vital step in many functional processes, including endoplasmic reticulum-to-Golgi vesicle trafficking13,49 and recycling of the SVs within the mechanism of neuronal communication11. In the present study, we have examined the structural principles underlying the αS-induced interactions and assembly of SVs by characterizing the membrane-binding properties of two mutational variants of αS linked with familial PD. This analysis reveals that two key regions of the protein, namely the N-terminal membrane-anchor (residues 1 to 25) and the central segment of the sequence (residues 65-97, having significant overlap with the NAC region), have independent membrane-binding properties and therefore are not only able to interact with a single SV, but can also simultaneously bind to two different vesicles ( Fig. 3a) thereby promoting their interaction and assembly as shown in vitro and in vivo 14,15,16. The resulting double-anchor mechanism explains why the deletion of the segment 71-82 in the NAC region of αS or the impairment of the membrane affinity of the N-terminal anchor region of the protein severely affects in vivo vesicle clustering as shown in Saccharomyces cerevisiae 16. This mechanism also provides a structural explanation for the suggested loss of function caused by A30P, which is associated with an impairment of vesicle clustering by αS A30P as observed both in vitro and in vivo 14,16, as well as for the functional regulation proposed to occur via the phosphorylation of serine 87 (ref. 50). In particular, by introducing a negative charge in the region 65-97, the phosphorylation of S87, which has been identified both in functional contexts and in the case of synucleinopathies, has similar effects to those of the K80E mutation in αS Sw.

We tested this molecular mechanism by engineering the mutational variant αS Sw, which was designed to enhance the probability of αS binding simultaneously to two different vesicles. Our studies of this variant have provided strong evidence in support of the proposed mechanism by showing that enhanced exposure of the central region, spanning residues 65-97 in the membrane-bound state of αS, promotes more strongly the clustering of SVs purified from rat brain ( Fig. 5) and the assembly and fusion of DOPE:DOPS:DOPC SUVs ( Fig. 4). It has previously been suggested that a broken α-helix structural topology of αS22,51, which is a conformation that αS adopts on binding to detergent micelles, could play a role in vesicle-vesicle interactions stimulated by αS48,52. The present study, however, shows experimentally that the underlying mechanism by which αS mediates the interactions between lipid vesicles relies on the balance between ordered (membrane-bound) and disordered (membrane-detached) conformational states of the region spanning residues 65-97 of the protein. Perturbing this balance, as we have done rationally with αS Sw, or on alteration of the expression levels of αS, can dramatically affect its ability to promote the vesicle assembly in vivo leading to defects in the regulation of vesicle trafficking11,13,49,53,54,55,56,57.

Other studies also suggest that αS could act as a molecular chaperone for the formation of SNARE complexes, which appears to result from the direct interaction between αS and synaptobrevin 2 at the surface of SVs14,58. Such an interaction was shown to be independent of the NAC region, suggesting that this region has no direct functional role in this particular process59. The present data, however, reveal that the NAC region is not only involved in αS aggregation, as extensive evidence has previously indicated4,60,61, but also has a specific role in a key molecular mechanism associated with the normal function of αS. This study provides evidence that the membrane affinity of the NAC region of αS is finely tuned to ensure an optimal degree of local detachment from the membrane surface to enable binding to occur between different vesicles. The present finding that, by perturbing this fine tuning through the design of the αS Sw variant, it is possible to promote stronger interactions between vesicles ( Figs 3 and 5) indicates that the exposure of the region 65-97 in the vesicle-bound state of αS is crucial for the physiological mechanism of SVs clustering and, at least in the case of αS Sw, has more relevance than the local membrane-binding affinity of this region, which in this variant is reduced as a result of the K80E mutation. The selection toward sequence properties of αS that enable the detachment of the amyloidogenic NAC region from the vesicle surface to favour the functional mechanism described in this study, however, can also lead to aberrant behaviour, as these conformational states are particularly vulnerable to self-association leading to αS aggregation at membrane surfaces11,62,63,64,65. Taken together, these findings provide therefore a new mechanistic link between functional and pathological roles of αS.

αS purification

αS WT was expressed and purified in Escerichia coli using a pT7-7 plasmid in which αS gene is under the control of the phage T7 RNA polymerase promoter. BL21 (DE3)-gold competent cells (Agilent Technologies, Santa Clara, USA) were transformed with this plasmid using heat-shock and subsequently grown in an isotope-enriched M9 minimal medium containing 1 g l −1 of ¹⁵N ammonium chloride, 2 g l −1 of ¹³C-glucose and 100 μg ml −1 ampicillin (Sigma-Aldrich, St Louis, USA) to produce uniformly ¹⁵N and/or ¹³C labelled αS samples. Cell growth was carried out at 37 °C under constant shaking at 250 rpm to an OD600 of 0.6. Subsequently the expression of the protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C for 4 h and cells were then harvested by centrifugation at 6,200 g (Beckman Coulter, Brea, USA). The cell pellets were resuspended in lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA and EDTA-free complete protease inhibitor cocktail tablets obtained from Roche, Basel, Switzerland) and lysed by sonication. The cell lysate was centrifuged at 22,000 g for 30 min to remove cell debris and the supernatant was then heated for 20 min at 70 °C and subsequently centrifuged at 22,000 g to precipitate the heat-sensitive proteins. Subsequently streptomycin sulfate was added to the supernatant to a final concentration of 10 mg ml −1 to stimulate DNA precipitation. The mixture was stirred for 15 min at 4 °C followed by centrifugation at 22,000 g. Then, ammonium sulfate was added to the supernatant to a concentration of 360 mg ml −1 to precipitate the protein. The solution was stirred for 30 min at 4 °C and centrifuged again at 22,000 g. The resulting pellet was resuspended in 25 mM Tris-HCl, pH 7.7 and dialyzed against the same buffer to remove salts. The dialyzed solutions were then loaded onto an anion exchange column (26/10 Q sepharose high performance, GE Healthcare, Little Chalfont, UK) and eluted with a 0 to 1 M NaCl step gradient. αS was eluted at ∼300 mM NaCl and then further purified by loading onto a size-exclusion column (Hiload 26/60 Superdex 75 preparation grade, GE Healthcare, Little Chalfont, UK). All the fractions containing the monomeric protein were pooled together and concentrated by using Vivaspin filter devices (Sartorius Stedim Biotech, Göttingen, Germany). The purity of the aliquots after each step was analysed by SDS-PAGE and the protein concentration was determined from the absorbance at 275 nm using an extinction coefficient of 5,600 M −1 cm −1.

To express and purify the mutational variants of αS (αS A30P, αS E46K and αS Sw), we employed the same experimental procedure as used in the case of αS WT. Single point mutations of the αS WT plasmid were obtained using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Inc., Ipswich, USA). Table 1 reports the primers employed to obtain the plasmids of αS A30P, αS E46K and αS Sw.

Preparation of synaptic-like SUVs

SUVs containing a molar ratio of 5:3:2 of DOPE:DOPS:DOPC (Avanti Polar Lipids, Inc., Alabaster, USA) were prepared from chloroform solutions of the lipids as described previously15,28. Briefly, the lipid mixture was evaporated under a stream of nitrogen gas and then dried thoroughly under vacuum to yield a thin lipid film. The dried thin film was re-hydrated by adding aqueous buffer (20 mM sodium phosphate, pH 6.0) at a concentration of 15 mg ml −1 (1.5%) and subjected to vortex mixing. In all NMR experiments described in this paper SUVs were obtained by using several cycles of freeze-thawing and sonication until the mixture became clear15,28. In the particular case of CEST experiments, SUVs at a concentration of 0.06% (0.6 mg ml −1) were mixed with αS samples after sonication. For ssNMR studies αS was added to the SUV mixtures up to a molar ratio of 1:65 protein:lipid. The mixtures were then pelleted at 300,000 g for 30 min at 4 °C (Beckman Coulter Optima TLX Inc. Brea, USA) by using a TLA 100.3 rotor. Subsequently the SUV-αS samples were transferred into 3.2 mm Zirconia XC thin-walled MAS rotors for ssNMR experiments. For STED and cryo-EM imaging experiments, as well as for DLS, DOPE:DOPS:DOPC SUVs were prepared by extrusion through membranes with a 50 nm pore diameter (Avanti Polar Lipids, Inc) after re-hydration in 20 mM sodium phosphate (pH 6.0) at a concentration of 1.0 mg ml −1 (0.1%).

Purification of SVs from rat brain

SVs composed of phospholipid molecules (amounting to 30% of their composition), proteins (58%) and cholesterol (12%)44,45 were purified from brains of rat provided by Charles River Laboratories (Animal work was approved by the Named Animal Care and Welfare Officer (NACWO) and the Ethics Review Committee of the University of Cambridge). Rat brains were homogenized in 4 mM HEPES and 320 mM sucrose buffer using protease inhibitors via ten strokes at 900 r.p.m. in a glass-Teflon homogenizer (Wheaton, Millville, USA)44,45. All steps were carried out at 4 °C or in ice. The homogenates were centrifuged at 1,000 g for 10 min and the supernatants were collected and further centrifuged at 15,000 g for 15 min. The supernatants from the second centrifugation (Sup-2) were stored at 4 °C. The pellets from the second centrifugation, which contained the synaptosomes, were lysed using ice-cold water by applying three strokes at 2,000 r.p.m. Subsequently, HEPES buffer solutions containing protease inhibitors were added to the lysates and the resulting solutions were centrifuged at 17,000  g for 15 min, and the supernatant was combined with the Sup-2 supernatants. The resulting mixtures were centrifuged at 48,000 g for 25 min and the supernatants were loaded onto a 0.7 M sucrose cushion and centrifuged at 133,000 g for 1 h. The bottom half of the sucrose cushion was pooled and centrifuged at 300,000 g for 2 h. The pellets were resuspended in buffer (100 mM Tris-HCl, pH 7.4, 100 mM KCl) and loaded onto a Sephacryl S-1000 size-exclusion chromatography column (100 × 1 cm) resulting in a distinctive peak of the SVs44,45. The SVs were then stained against specific SV antibodies, including synaptotagmin 1, synaptobrevin 2, by western blot44,45. To perform dSTORM analyses, the SVs were incubated with a primary antibody (dilution 1:1000) that specifically recognizes synaptotagmin 1 (105103, Synaptic Systems, Göttingen, Germany) and a secondary antibody (dilution 1:100) fluorescently labelled with Atto 647N (40839, Sigma-Aldrich, St Louis, USA).

CEST experiments

We employed CEST measurements28,36,37,38,40 to gain a deeper understanding of the equilibrium between membrane unbound and membrane-bound states of αS. In the study of αS-SUV interactions, CEST shows enhanced characteristics compared to standard heteronuclear correlation spectroscopy, including a significantly higher sensitivity at low lipid:protein ratios, conditions under which protein or lipid aggregation can be minimized64. The resulting NMR signals enable the interaction between αS and the membrane surface to be probed without interference from additional factors that may influence the transverse relaxation rates of the protein resonances36,37,38,40. In the CEST experiments employed here, a continuous weak radiofrequency field (either 350 or 170 Hz) was applied off-resonance (up to ±28 kHz) in the ¹⁵N channel, thereby saturating the broad spectroscopic transitions in the bound (undetectable) state but leaving the resonances of the free (detectable) state virtually unperturbed36,37,38,40. The saturation of the bound state was then transferred to the free state via chemical exchange, resulting in the attenuation of the intensities of the observable resonances in the visible unbound state. By carrying out a series of experiments at various offsets, it was possible to obtain a map of the strength of the interactions between the low (unbound αS) and high (SUV-bound αS) molecular weight species at a residue specific resolution.

Solution-state NMR experiments were carried out at 10 °C on Bruker spectrometers operating at ¹H frequencies of 700 MHz equipped with triple resonance HCN cryo-probes. CEST experiments were based on ¹H- ¹⁵N HSQC experiments by applying constant wave saturation in the ¹⁵N channel. Since we aimed at probing the exchange between monomeric αS (having sharp resonances) and αS bound to SUVs (having significantly broader resonances), a series of large offsets was employed (−28, −21, −14, −9, −5, −3, −1.5, 0, 1.5, 3, 5, 9, 14, 21 and 28 kHz), resulting in CEST profiles of symmetrical shapes ( Supplementary Fig. 1)28,36,37. An additional spectrum, saturated at −100 kHz, was recorded as a reference. The CEST experiments were recorded using a data matrix consisting of 2,048 (t ₂, ¹H) × 220 (t ₁, ¹⁵N) complex points. Assignments of the resonances in ¹H- ¹⁵N HSQC spectra of αS WT were derived from our previous studies28 whereas assignments of the mutational variants employed in this work were obtained by a standard combination of triple resonance solution NMR spectra66.

MAS measurements

MAS provides complementary information to CEST as it allows the protein resonances in the vesicle-bound state, which is inaccessible to solution-state NMR, to be probed directly. MAS experiments were carried out on a 16.85 T Bruker Spectrometer with a 3.2 mm E Free probe. DARR experiments42 were performed at a MAS rate of 10 kHz using a series of different mixing times (20, 50, 100, 200 and 500 ms), and the spectra were acquired at −19 and 4 °C (the latter for control experiments only) using a 1 ms contact time. INEPT spectra41 were measured at 4 °C using a MAS rate of 10 kHz. Pulse widths were 2.5 μs for ¹H and 5.5 μs for ¹³C, and ¹H TPPM decoupling was applied at ωRF/(2π)=71.4-100 kHz (ref. 28).

Cryo-EM measurements

All samples used in cryo-EM measurements were incubated, with or without αS (200 μM), for 12 h at 298 K using fresh preparations of DOPE:DOPS:DOPC SUVs at a concentration of 0.05%. After incubation cryo-EM grids were prepared by vitrifying the sample solutions using aliquots of 2 μl and a Vitrobot Mark IV (FEI, Hillsboro, USA) at a relative humidity of 100%. The samples were loaded on a glow-discharged Quantifoil Copper 300 mesh R2/2 grids (Quantifoil Micro Tools GmbH, Germany) and blotted with filter paper for 2.5 s to leave a thin film of solution. The blotted samples were plunged into liquid ethane and stored under liquid nitrogen before imaging. Samples were examined using a Philips CM200 FEG electron microscope operating at 200 kV (FEI, Hillsboro, USA), using a Gatan 626 cryo-holder (Gatan, Pleasantos, USA) cooled with liquid nitrogen to temperatures below −180 °C. Digital images were acquired on a TVIPS FC415 CCD camera using the EMMENU 4 software package (TVIPS, Munich, Germany).

STED microscopy

STED imaging67,68 was carried out by fluorescently labelling the DOPE:DOPS:DOPC SUVs with 2% of fluorescently labelled DOPE (ATTO 647N DOPE, ATTO-TECH, USA). STED microscopy allows the diffraction limit in optical microscopy (∼200 nm) to be overcome69 and imaging was performed on a home-built pulsed STED microscope69 using a single titanium-sapphire oscillator centred at λ STED=765 nm (Ti:S, Mai Tai HP, Spectra-physics, Santa Clara, USA) to generate the STED beam, which was subsequently split into two using a half-plate and a polarization beam splitter. Of these two beams, the one transmitted was focused onto a photonic crystal fibre (FemtoWhite, NKT Photonics, Cologne, Germany) to produce white light radiation. From this light, an excitation beam, centred at λ Exc=640 nm, was extracted using a bandpass filter (637/7 BrightLine HC, Semrock, NY, USA) and coupled into a 30 m long polarization maintaining single-mode fibre (PM630-HP, Thorlabs, Newton, UK). The reflected STED beam was passed through a 50 cm long glass block of SF66 and a 100 m long polarization maintaining single-mode fibre (PM-S630-HP, Thorlabs, Newton, UK) to stretch the pulse duration to ∼100-200 ps. In addition, the STED beam was converted into a so called donut beam by a spatial light modulator (X10468−02, Hamamatsu Photonics, Hamamatsu City, Japan). The excitation and STED beams were recombined with a dichroic mirror (T735spxr, Chroma, Bellow Falls, USA) and detected using a commercial point-scanning microscope (Abberior Instruments, Göttingen, Germany) comprising of a microscope frame (IX83, Olympus, Shinjiuku, Japan), a set of galvanometer mirrors (Quad scanner, Abberior Instruments, Göttingen,, Germany) and a detection unit. The beams were focused onto the sample by a × 100/1.4 NA oil immersion objective lens (UPLSAPO 100XO, Olympus, Göttingen, Germany) and images were acquired by raster scanning the beams across the sample using the Inspector software (Andreas Schönle, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). We used a field of view of 30 × 30 μm ² with a pixel size of 15 × 15 nm ² and a pixel dwell time of 20 μs. Fluorescence photons emerging from the sample were collected by the microscope objective lens, de-scanned by the galvanometer mirrors, focused onto a pinhole and detected using an avalanche photodiode (SPCM-AQRH, Excelitas Technologies. Waltham, USA). The laser powers, measured at the objective back aperture, were ca. 20 μW and 150 mW for the excitation beam and for the STED beam, respectively.

Analysis of STED images for vesicle size measurement

Vesicle sizes were estimated from STED images by using in-house Matlab scripts ( Supplementary data 1). First, images of isolated vesicles were identified and analysed using a fitting based on a 2D Gaussian function, by convolving the images with a Gaussian filter whose dimensions match the extension of the expected STED point spread function. The centres of the vesicles were identified by finding local maxima of the convolved images, excluding the local maxima corresponding to fused vesicles by means of a threshold applied on the peak intensities. A different fitting procedure was optimized in the case of assembled vesicles that appear as hollow shapes in the STED images, as for a vesicle larger than the lateral resolution of the STED microscope the number of dye molecules probed increases on the edge of the shell. To estimate the size of the clusters and their relative number compared to the non-fused vesicles, all the vesicles appearing as fused were fitted by annular functions having a Gaussian radial profile (amplitude, position, radius and offset) using a nonlinear least squares approach.

Direct stochastic optical reconstruction microscopy

Super-resolution imaging was performed using dSTORM microscopy with a Nikon Eclipse TE 300 inverted wide-field microscope using a × 100, 1.49-N.A total internal reflection fluorescence46 objective lens (Nikon Ltd., Kingston upon Thames, UK). The vesicle and αS samples were adhered to a glass coverslip coated in poly-L-lysine (P4707, Sigma-Aldrich, St Louis, USA) before photoswitching buffer solution was added, consisting of 100 mM mercaptoethylamine (MEA) in phosphate buffered saline (PBS, pH 8.2). For imaging, a laser emitting at a wavelength of 640 nm was used (Toptica Photonics AG, Graefelfing, Germany) for excitation of the Atto 647N dye. A 405 nm laser (Mitsubishi S3 Electronics Corp., Tokyo, Japan) was used as the reactivation source, which was only turned on when the number of active fluorophores in the field of view was visibly reduced. Imaging was performed under total internal reflection fluorescence illumination conditions, ensuring that the exact centre of the field of view was illuminated. The field of view covered 1,997 × 1,997 camera pixels, corresponding to an area on the sample of ∼20 × 20 μm ². 10,000 fluorescence frames were recorded, each corresponding to an exposure time of 10 ms; the latter was matched to be in the range of the average 'on' time of the fluorescent dyes. The fluorescence light in the detection path was filtered and imaged with an Ixon DV887 ECS-BV EM-CCD camera (Andor, Belfast, UK). The image analysis was performed using frames 1000 to 10 000 in each sequence. From each image stack, a reconstructed dSTORM image was generated using the open-source rapidSTORM software ( Supplementary Data 2) developed in house using MATLAB (The MathWorks, Inc.).

Dynamic light scatterning

DLS measurements of vesicle size distributions were performed using a Zetasizer Nano ZSP instrument (Malvern Instruments, Malvern, UK) with backscatter detection at a scattering angle of 173°. The viscosity (0.8882 cP) and the refractive index (1.330) of water were used as parameters for the buffer solution, and the material properties of the analyte were set to those of the lipids (absorption coefficient of 0.001 and refractive index of 1.440). SUVs were used at a concentration of 0.05% in these measurements and the experiments were performed at 25 °C. The acquisition time for the collection of each dataset was 10 s and accumulation of the correlation curves was obtained using 10 repetitions. Each measurement was repeated 10 times to estimate standard deviations and average values of the centres of the size distributions ( Supplementary Fig. 11).

Modelling

Schematic representations of αS bound to SUVs were obtained by using molecular dynamics (MD) simulations in implicit solvent. The structure of αS in the double-anchor mechanism ( Fig. 3a) were obtained by starting from the model of membrane-bound conformation αS characterized by an elongated helix (residues 1 to 97) with a disordered C-terminal region (residues 98-140) which was part of the ensemble characterized previously28. Atomic coordinates (in Cartesian space) of the N-terminal anchor were harmonically restrained to maintain a fixed position whereas the region spanning residues 65-97 was restrained in the alpha-helical conformation. A constant force along the membrane normal was applied to this region to extend it toward the second vesicle (up in the Fig. 3a). The reminder of the protein (residues 26-59 and 98-140) was allowed to relax under the Newtonian laws of motions during the MD simulations. Curved vesicle surfaces were generated by starting from atomic models of DOPE:DOPS:DOPC bilayers and by generating roto-translations that imposed a spherical symmetry with a radius of 25 nm.

Data availability

Data supporting the findings of this study are available within the article and its Supplementary Information Files and from the corresponding author on request.

How to cite this article: Fusco, G. et al. Structural basis of synaptic vesicle assembly promoted by α-synuclein. Nat. Commun. 7:12563 doi: 10.1038/ncomms12563 (2016).

This research was supported by Parkinson's UK (H-1103 to G.F. and C.M.D.), The Wellcome Trust (104933/Z/14/Z to A.D. and 089703/Z/09/Z to C.F.K.), the UK Medical Research Council (MR/N000676/1 to G.F., M.V., C.M.D., A.D. and MR/K015850/1 and MR/K02292X/1 to C.F.K.) and the UK Engineering and Physical Sciences Research Council (EP/H018301/1 to C.F.K.).

Author notes

Pierre Mahou

Present address: Present address: Laboratory for Optics and Biosciences, Ecole Polytechnique, Palaiseau, 91128 Cedex, France,

Authors and Affiliations

1. Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK

Giuliana Fusco, Ana Rita Costa, Michele Vendruscolo & Christopher M. Dobson

2. Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK

Tillmann Pape & Alfonso De Simone

3. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK

Amberley D. Stephens, Pierre Mahou, Clemens F. Kaminski & Gabriele S. Kaminski Schierle

4. Department of Chemistry & Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, 55455, Minnesota, USA

Gianluigi Veglia

Contributions

G.F., C.M.D. and A.D. designed the research. G.F., G.S.K., G.V., C.M.D. and A.D. designed the experiments. G.F., T.P., A.D.S., P.M., A.C. and A.D. performed the experiments. G.F., T.P., A.D.S., P.M., C.F.K., G.S.K., M.V., G.V., C.M.D. and A.D. analysed the data. G.F., C.M.D. and A.D., wrote the manuscript. All authors revised the manuscript critically and approved the final version.

Corresponding authors

Correspondence to Christopher M. Dobson or Alfonso De Simone.

Competing interests

The authors declare no competing financial interests.

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
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# data[].attributes.attachment_link.title
Structural basis of synaptic vesicle assembly promoted by α-synuclein - Nature Communications

# data[].attributes.attachment_link.description
α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly.

# data[].attributes.body
Add SUVs -> CD spectrum dips to around 220 nm, characteristic of α helix

Affinity is 3x higher for POPG than DOPC/DOPS/DOPE lipids

# data[].attributes.body
ssNMR of αS aimed to determine:

Structure and dynamics of αS bound to synaptic-like vesicles

Topological properties

Molecular basis for the membrane affinity

# data[].attributes.headline
Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

# data[].attributes.subject
Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

# data[].attributes.attachment_link.description
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# data[].attributes.body
αS binds to SUV membranes (mimics synaptic vesicles)

# data[].attributes.body
Imperfect repeats of KTKEGV motif throughout the 1-70 region --> positively-charged, disordered N terminus

Negatively-charged C terminus

# data[].attributes.body
PREs provide long-range topological restraints

# data[].attributes.body
MAS ssNMR can be used to study large proteins and membrane proteins

# data[].attributes.body
Use different experiments for rigid domains and flexible domains (give separate spectra)

# data[].attributes.headline
Magic angle spinning (MAS): 54.74o relative to the magnetic field

# data[].attributes.subject
Magic angle spinning (MAS): 54.74o relative to the magnetic field

# data[].attributes.body
Fast rotation around MAS averages out the DC and CSA effects (which was causing fast relaxation and line broadening) --> sharper resonance lines and higher resolution in the NMR spectra

# data[].attributes.headline
There is extensive broadening due to chemical shift anisotropy (CSA) and dipolar coupling (DC) in solids

# data[].attributes.subject
There is extensive broadening due to chemical shift anisotropy (CSA) and dipolar coupling (DC) in solids

# data[].attributes.body
As we go from fast tumbling in solution to the solid state (no tumbling), NMR lines broaden significantly up to total disappearance in standard solution NMR

# data[].attributes.headline
Increasing molecular weight leads to slower molecular tumbling and faster relaxation 

# data[].attributes.subject
Increasing molecular weight leads to slower molecular tumbling and faster relaxation 

# data[].attributes.body
Time dependent signal decays faster --> poor resolution

# data[].attributes.body
The reorientation of the molecules with respect to the magnetic field and the subsequent interaction of magnetic dipoles and chemical shift anisotropy gives rise to time dependent field fluctuations

# data[].attributes.headline
Use ssNMR to study disordered regions in membrane proteins 

# data[].attributes.subject
Use ssNMR to study disordered regions in membrane proteins 

# data[].attributes.headline
Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

# data[].attributes.subject
Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

# data[].attributes.body
In DNAJB6 we found a 10 residue segment within a 70-residue long disordered region that forms a helix which interacts with the J domain --> huge implications for function as it inhibits binding of the partner protein and keeps the protein in an auto-inhibited form

# data[].attributes.body
Combining chemical shifts (report on secondary structure) with PREs (reports on topology of sec. structure elements) is a powerful tool to investigate residual structure in IDPs

# data[].attributes.body
PRE was used to determine the topology of the DNAJB6 helix

If the spin label is attached N-terminally to helix 5, PRE effects are observed in helix 2

If the spin label is attached C-terminally to helix 5, PRE effects are observed in helix 3

# data[].attributes.headline
Topology of secondary structure elements

# data[].attributes.subject
Topology of secondary structure elements

# data[].attributes.body
Attach labels with lone electrons (paramagnetic species) to Cys (can engineer) --> increases R₂ of nearby groups --> broader peaks

We can attach spin labels at various places on a protein and see which regions are affected --> allows us to determine the topology of secondary structure elements

# data[].attributes.body
Can still apply 3D NMR assignment method to IDPs because peaks are very sharp

# data[].attributes.headline
From chemical shifts to residual secondary structures

# data[].attributes.subject
From chemical shifts to residual secondary structures

# data[].attributes.body
e.g. DNAJB6 is a key component of the chaperone network involved in the Hsp40-Hsp70 cycle (important in protection from many neurodegenerative diseases)

The protein can be divided in two structured N, C-term domains linked by large disordered regions

The mixed structural nature of this protein is accurately accounted by chemical shifts

# data[].attributes.headline
Measuring and assigning chemical shifts of IDPs

# data[].attributes.subject
Measuring and assigning chemical shifts of IDPs

# data[].attributes.body
It is possible to investigate the residual secondary structure in IDPs by measuring 2D and 3D NMR spectra tailored to assign the backbone chemical shifts (e.g. HNCA, HNcoCA, CBCAcoNH, HNCACB, HNCO, HNcaCO) and by using these data in methods such as Talos, δ2D

# data[].attributes.headline
Determination of Secondary Structure Populations in Disordered States of Proteins Using Nuclear Magnetic Resonance Chemical Shifts

# data[].attributes.subject
Determination of Secondary Structure Populations in Disordered States of Proteins Using Nuclear Magnetic Resonance Chemical Shifts

# data[].attributes.body
Chemical shifts are sensitive to the local backbone structure

Since chemical shifts are averaged the secondary structure element does not have to be present 100% of the time

Using this principle we can take advantage of the chemical shifts to estimate propensities/populations of helices, strands, poly-proline helices, β-turns even if they are not fully formed

Many programs/servers exist that can do this e.g. Talos, δ2D

# data[].attributes.body
Proteins exist in continuum of functional dynamics: from mainly ordered proteins, proteins with folded domains and disordered regions (IDRs) or completely disordered proteins (IDPs)

At least 30% of the proteome is disordered and often the disordered regions are those that are functionally important

Current AI based tools (like AlphaFold) completely fail to characterize IDRs/IDPs

# data[].attributes.headline
Chemical shifts can be used to determine residual secondary structure 

# data[].attributes.subject
Chemical shifts can be used to determine residual secondary structure 

# data[].attributes.body
Different backbone conformations --> different chemical shifts

Similarly to the random coil, we can predict chemical shifts associated with secondary structure elements

By comparing measured and predicted chemical shifts it is therefore possible to assess the amount of secondary structure in a protein (folded or disordered)

Due to the separation in the distributions of chemical shifts from the different secondary structures

# data[].attributes.body
CPMG is sensitive to dynamics ranging in the μs to ms timescale

CPMG is most effectively used when two conformations are in intermediate exchange

CPMG provides three major data: Populations, kex and Δω

# data[].attributes.body
As ligands change, […], the energy landscape and the populations of the accessible states change in response. Thus, the dynamic energy landscape efficiently funnels the enzyme through its catalytically competent conformations along a preferred kinetic path, where the number and heights of the energetic barriers between consecutive conformations have been minimized.

# data[].attributes.headline
The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis

# data[].attributes.subject
The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis

# data[].attributes.headline
Conservation of μs−ms Enzyme Motions in the Apo- and Substrate-Mimicked State

# data[].attributes.subject
Conservation of μs−ms Enzyme Motions in the Apo- and Substrate-Mimicked State

# data[].attributes.body
14 kDa enzyme

Catalyses transphosphorylation of single-stranded RNA

No cofactors or metal ions

Rate-limiting step in the catalysis is product release (k cat ≈ k off)

# data[].attributes.body
Chemical exchange in the ms timescale can be effectively studied by CPMG NMR experiments even if the population of the second state is <5%

# data[].attributes.body
State B is never directly detected

# data[].attributes.body
ΔωB :  chemical shift of state B, relates to the structure of the excited state

# data[].attributes.body
k AB: Forward rate, k BA: Backward rate

Temperature dependence gives size of barrier

# data[].attributes.body
pB, k AB, k BA, ΔωB can give information on the mechanism

# data[].attributes.body
pB: Population of state B, relates to equilibrium position Ke, Kd

Temperature dependence of Ke or Kd (Van’t Hoff analysis) gives ΔΗ, ΔS

# data[].attributes.headline
Designing mutants with targeted properties 

# data[].attributes.subject
Designing mutants with targeted properties 

# data[].attributes.headline
Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

# data[].attributes.subject
Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

# data[].attributes.attachment_link.readable_text
Letter

Published:

Guillaume Bouvignies 1,2,3 na1,

Pramodh Vallurupalli 1,2,3 na1,

D. Flemming Hansen 1,2,3,

Bruno E. Correia 4,5,

Oliver Lange 4,

Alaji Bah 6,

Robert M. Vernon 4,6,

Frederick W. Dahlquist 7,

David Baker 4 &

...

Lewis E. Kay 1,2,3,6

Show authors
Nature 477, 111-114 (2011) Cite this article

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Abstract

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers)1,2,3. However, a rigorous understanding of the relation between a protein's structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules4 and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the 'invisible', excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta7 model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein's function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

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The structural ensembles of the L99A excited state and L99A,G113A,R119P T4L have been deposited with PDB (accession codes 2LCB, 2LC9).

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Acknowledgements

We thank J. Forman-Kay for providing laboratory space, and J. Forman-Kay and T. Alber for discussions. G.B. acknowledges The European Molecular Biology Organization and The Canadian Institutes of Health Research (CIHR) for postdoctoral fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. This work was supported by the CIHR and the Natural Sciences and Engineering Research Council of Canada, with computations performed on the GPC supercomputer at the SciNet HPC Consortium.

Author information

Author notes

Guillaume Bouvignies and Pramodh Vallurupalli: These authors contributed equally to this work.

Authors and Affiliations

1. Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada

Guillaume Bouvignies, Pramodh Vallurupalli, D. Flemming Hansen & Lewis E. Kay

2. Department of Biochemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada

Guillaume Bouvignies, Pramodh Vallurupalli, D. Flemming Hansen & Lewis E. Kay

3. Department of Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada

Guillaume Bouvignies, Pramodh Vallurupalli, D. Flemming Hansen & Lewis E. Kay

4. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA

Bruno E. Correia, Oliver Lange, Robert M. Vernon & David Baker

5. Program in Computational Biology, Instituto Gulbenkian de Ciência, P-2780-156 Oeiras, Portugal

Bruno E. Correia

6. Hospital for Sick Children, Program in Molecular Structure and Function, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada

Alaji Bah, Robert M. Vernon & Lewis E. Kay

7. Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, 93106, California, USA

Frederick W. Dahlquist

Contributions

G.B., P.V. and D.F.H. made samples. G.B., P.V., D.F.H. and L.E.K. designed and performed all NMR experiments. G.B., P.V., B.E.C., O.L. and R.M.V. performed structure calculations. G.B., P.V. and A.B. carried out initial crystallization trials. F.W.D, D.B., G.B., P.V. and L.E.K. designed experiments and wrote the paper.

Corresponding author

Correspondence to Lewis E. Kay.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

The file contains Supplementary Figures 1-10 with legends, Supplementary Tables 1-9 and additional references. (PDF 4402 kb)

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Bouvignies, G., Vallurupalli, P., Hansen, D. et al. Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477, 111-114 (2011). https://doi.org/10.1038/nature10349
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DOI: https://doi.org/10.1038/nature10349

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Solution structure of a minor and transiently formed state of a T4 lysozyme mutant - Nature

# data[].attributes.attachment_link.description
The function of a protein depends critically on structural dynamics, and on the nature of the transient conformation intermediates that the molecule can adopt. These transients can be elusive and therefore hard to characterize.

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'Invisible' conformational state: 3% (298K), 1ms lifetime

# data[].attributes.body
PX structures of T4 lysozyme cannot explain how the ligand enters the binding pocket

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An S/T motif controls reversible oligomerization of the Hsp40 chaperone DNAJB6b through subtle reorganization of a β sheet backbone

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An S/T motif controls reversible oligomerization of the Hsp40 chaperone DNAJB6b through subtle reorganization of a β sheet backbone

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A Transient and Low-Populated Protein-Folding Intermediate at Atomic Resolution

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A Transient and Low-Populated Protein-Folding Intermediate at Atomic Resolution

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CPMG can derive information on a per-residue level about lowly-populated states (only few % in solution) that would not otherwise be detected

1. Thermodynamics

2. Kinetics

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Does not have to be 2-state!