α-synuclein-lanthanide metal ions interaction: binding sites, conformation and fibrillation
© Bai et al. 2016
Received: 11 October 2015
Accepted: 15 January 2016
Published: 3 February 2016
The pathological hallmark of Parkinson’s disease is the deposition of cytoplasmic neuronal inclusions termed Lewy bodies. The major component of Lewy bodies is amyloid fibrils of α-synuclein. To investigate what causes α-synuclein aggregation is essential to understand its pathological roles in Parkinson’s disease. Various metal ions, including iron and copper, have been implicated in the pathogenesis of Parkinson’s disease. Divalent metal ions can regulate α-synuclein fibrillation rate, however, few studies have been performed to investigate how trivalent metal ions interact with α-synuclein and their effect on α-synuclein fibrillation. The study of the interaction between divalent and trivalent metal ions with α-synuclein is of vital importance to realize the mechanism of α-synuclein fibrillation.
Here we used nuclear magnetic resonance spectroscopy to determine the trivalent metal ions (lanthanides) binding sites in α-synuclein. We found that lanthanide metal ions not only bind non-specifically to the C-terminal domain of α-synuclein, but also transiently interact with residues contain carboxyl groups in the N-terminal and NAC regions, the latter binding sites were not found for divalent cations. In addition, lanthanide ions bound α-synuclein exhibits slower conformational exchange rate. Compare to divalent cations, lanthanide ions accelerate α-synuclein fibrillation much faster.
We identified the lanthanide metal ions binding sites in α-synuclein and found a hierarchal effect for lanthanide ions binding to α-synuclein, driven by the interaction with aspartic acids and glutamic acids residues. Lanthanide ions binding also induced conformational dynamics change of α-synuclein. Compared to divalent cations, lanthanide metal ions significantly accelerated α-synuclein fibrillation, possibly due to the different inherent properties such as charge, binding sites and coordination modes.
Keywordsα-synuclein (αS) Lanthanide metal ions Binding sites Fibrillation
Parkinson's disease (PD) is a common neurodegenerative disease of the population over 65 . The histological hallmark of Parkinson’s disease is the selective missing of dopaminergic neurons in the substantia nigra pars compacta. Intraneuronal deposits of fibrillar and misfolded proteins called the Lewy bodies appear in the affected brain areas. The main component of the Lewy bodies is α-synuclein (αS) aggregates [1–3]. Investigating what causes αS aggregation is important to understand its pathological roles in PD.
In aqueous solution, monomeric αS is a natively unfolded protein with no apparent ordered secondary structure detectable by Far-UV Circular Dichroism (CD), Fourier transform infrared spectroscopy, or nuclear magnetic resonance (NMR) spectroscopy [4–6], but it forms β-sheet riched amyloid like fibrils under certain conditions [4, 7–9]. The N-terminal of αS exhibits a partially α-helical secondary structure upon binding with negatively phospholipid membranes and detergent micelles, while the C-terminus still remains dynamically unstructured [10–14]. αS has 140 amino acids with three distinct regions: the positively charged N-terminal region (residues 1–60); the hydrophobic NAC (non-amyloid β component) region (residues 61–95); and the highly negatively charged C-terminal region (residues 96–140) [15, 16]. The exposure of NAC region is considered as the main reason for αS fibrillation . αS has the structural properties of auto-inhibition fibrillation due to the long-range transient intra-molecular interaction between the N-terminus and the C-terminus, which protecting NAC region from exposure [17–20].
Increasing evidences have shown that altered metal homeostasis might be involved in the progression of neurodegenerative diseases. The possible involvement of heavy metals in the etiology of PD followed primarily from the results of epidemiological studies [21–24] and Lewy bodies component analysis in the parkinsonian substantia nigra [25, 26]. The potential link between metal ions and the PD related protein αS was observed in in vitro experiments. Recent studies show that the binding affinities of αS for diverse metal ions are different, but the binding sites are similar for the majority of metal ions. For divalent cations such as Fe2+, Mn2+, Co2+,Ni2+ and Ca2+, they all bound non-specifically to the C-terminal domain of αS [27–29]. But for Cu2+, it has high affinity to the αS N-terminus, and low affinity to the C-terminus [27, 30, 31]. No conformational change of αS was observed at low ionic concentration of various ions. At high concentration of metal ions, K+, Na+, Li+, Cs+, and Ca2+ has no effect on the unfolded structure of αS, but Mn2+, Cd2+, Mg2+ and Zn2+ induce a small increase in α-helix contents, and Cu2+, Co2+, Fe3+ and Al3+ induce more α-helix contents [32, 33].
Recently, studies have shown that lanthanide ions might affect the neuronal systems, but the toxicological behaviours were very complicated and the effects depended on a variety of factors [34–36]. With the increasing applications of lanthanides in industry, agriculture and medicine [37–41], particularly for the people who has long-term exposure in the electronics components industry or mining, public concern pays more and more attention on the toxicity of the lanthanides. In this study, we characterized the interaction between lanthanide metal ions (Ln3+) with recombinant human αS and the binding sites (region) were determined using NMR spectroscopy. We found that lanthanide metal ions accelerated αS fibrillation much faster than divalent cations in vitro. Based on the interaction information, we proposed the mechanism by which lanthanide metal ions accelerated αS fibrillation in vitro.
Results and discussion
Lanthanide metal ions binding sites determination
The intensity ratios of cross peaks in the presence (I) and absence of Lu3+ (I0) were also shown in Fig. 2d-f. When the molar ratio of Lu3+/αS decreased from 4/1 to 1/1, the signal intensity of residues located in the C-terminal region decreased, whereas small or no change was observed for the residues in the N-terminal and NAC regions. We also noticed that many cross peaks of C-terminal residues disappear when αS/Lu3+ ratio decreased further to 1/10 (Additional file 1: Figure S1), suggesting that binding with Lu3+ slows down αS conformational exchange rate.
We also used 1D 1H spectra to obtain information on the roles of αS aromatic side chains in lanthanide binding. The 1H NMR spectra of αS in D2O (6.3-7.5 ppm) comprised the side chains of different aromatic residues: Phe (F4, F94), Tyr (Y39, Y125, Y133, Y136) (Additional file 1: Figure S2 and Figure S3), and the signals were assigned according to previous reports . In the presence of low concentration lanthanide ions (Tb3+ and Dy3+), the Tyr signals intensity decrease significant, and with lanthanide ions (Tb3+ and Dy3+) concentration increasing, the Phe signals were further affected, suggesting lanthanide ions bound preferentially to C-terminus and might have transient weak interactions with N-terminal and NAC regions, consistent with 1H-15N HSQC spectra.
Many other metal ions and polyamines  also bind to the αS C-terminal region from residue 110 to 140. Other than the C-terminus, weaker binding sites involved residues 46–55 in the αS N-terminus was also reported for paramagnetic divalent cations such as Fe2+ and Co2+. For Cu2+, the specific binding sites were identified as residues Met1, Asp2, and His50 in the N-terminus [27, 30]. It was worth mentioning that the binding sites of Ln3+ involved residues different from those binding with other divalent cations [27, 28, 30]. Residues containing a carboxyl groups in the N-terminal and NAC regions also have transient weak interaction with lanthanide ions. The binding character difference might be due to the inherent properties of different metal ions, such as charge, ionic radii and coordination abilities. The lanthanide metal ions, which possess ionic radii similar to Ca2+, are assumed to behave very similar to Ca2+ in protein binding. Through NMR titration experiment, we found that Ca2+ binding sites located in the αS C-terminus (Additional file 1: Figure S4 and Figure S5). The chemical shift perturbations of C-terminal residues are much smaller in the presence of Ca2+ than that of Lu3+, suggesting that αS-Ca2+ binding is relatively weak. The pattern of the chemical shift perturbation was also different from that induced by lanthanides ions. Our observations suggest that although lanthanides ions have similar ionic radii to Ca2+, they show different binding properties with αS. Diamagnetic metal ion Al3+ was also reported as an effective promoter to αS fibrillation, and we also studied Al3+ binding sites in αS (Additional file 1: Figure S6 and Figure S7). We found no obvious chemical shift perturbations were observed in the presence of Al3+, and only large intensity attenuation of cross peaks were located in the αS C-terminus, which suggested that binding with Al3+ slows down αS conformational exchange rate. Such conformational exchange was also observed in the presence of high concentration of Lu3+ (Additional file 1: Figure S1).
Since the affinities for the Ln3+ with αS is similar, the different degree of broadening for Dy3+ and Tb3+ must be from slightly different paramagnetic properties: the total angular momentum quantum number J for Dy3+ is 15/2, which is a little larger than that for Tb3+ (J = 6), meanwhile the unpaired electron correlation time τe of Dy3+ is 0.3 × 10−12s, which is a little larger than that of Tb3+ (τe = 0.2 × 10−12s) . These different properties may explain why the intensity ratios of cross peaks in the presence of Dy3+ decrease more severely than that in the presence of Tb3+. The strong paramagnetic properties of lanthanide ions might have solvent PRE effect, which makes the cross peaks intensity ratios of most observed residues in the N-terminal and NAC region decreased to 0.4 or 0.6 (Fig. 3c, f).
As for diamagnetic ion (Lu3+), significant chemical shift perturbations were not observed in the N-terminal and NAC regions. It may be due to the fact that PRE is more sensitive to local structural perturbation than chemical shift, because paramagnetic relaxation enhancement is proportional to r−6, where r is the distance between paramagnetic ion and nucleus observed.
Lanthanide metal ions effects on αS conformation
The measured translational diffusion coefficients of dioxane and α-synuclein and the calculated hydrodynamic radius of α-synuclein at different concentration ratios of αS/Lu3+
CαS : CLu3+
Translational diffusion coefficients
Hydrodynamic radius RH (Å)
αS (methyl regoins from 0.87–0.56 ppm) D t × 10−11(m2/s)
Dioxane (3.65–3.53 ppm) D t × 10−10(m2/s)
1 : 0
5.802 ± 0.001
8.482 ± 0.004
30.99 ± 0.05
4 : 1
5.949 ± 0.004
8.610 ± 0.004
30.68 ± 0.03
2 : 1
5.823 ± 0.006
8.488 ± 0.004
30.90 ± 0.04
1 : 1
5.845 ± 0.004
8.575 ± 0.004
31.10 ± 0.03
1 : 2
5.939 ± 0.003
8.626 ± 0.004
30.79 ± 0.02
1 : 4
5.915 ± 0.003
8.694 ± 0.005
31.16 ± 0.02
Lanthanide metal ions effects on αS fibrillation
Many metal ions can accelerate αS fibrillation. The reason is very complex, and many factors involved in it. The different effects on αS fibrillation rate might be due to the inherent properties of different metal ions, such as binding sites, coordination modes. Positively charged metal ions neutralizing negatively charged αS, and resulting in stronger intermolecular association is also regarded as a reason for αS faster aggregation. Besides binding with αS C-terminus, lanthanide metal ions show very novel coordination modes, and residues contain the carboxyl groups in αS N-terminal and NAC regions that also have transient weak interaction with lanthanide ions, which is not reported in other studies of divalent metal ions interacting with αS. This specific interaction can disturb αS local conformation, and may be another reason for αS faster fibrillation. We also notice that many cross peaks of C-terminal residues disappear when αS/Lu3+ ratio decreased further to 1/10, suggesting that the presence of Lu3+ reduces the αS conformation exchange rate. Such conformational exchange reduction is also observed in the presence of Al3+. Slow αS conformational exchange rates make the expose hydrophobic residues contact long enough to form associated oligomers, which is crucial for protein fibrillation. When the conformational exchange rate is fast or almost the same as the bimolecular encounter rate, there is not enough time for molecules association occurring.
In summary, we identified the lanthanide metal ions binding sites in αS by employing the chemical shift perturbations and paramagnetic effects of these metal ions. In particular, we found a hierarchal effect for lanthanide ions binding to αS, driven by the interaction with specific residues, namely, aspartic acids, glutamic acids residues. Compared to divalent cations, lanthanide metal ions significantly accelerated αS fibrillation, possibly due to their different inherent properties such as charge, binding sites and coordination modes. The study here also suggest that binding induced change of conformational exchange dynamics provide a possible molecular mechanism to understanding αS fibrillation.
Protein expression and purification
Plasmids contained the coding sequence of α-synuclein were transformed into Escherichia coli strain BL21 (DE3) competent cells. Expression and purification of 15N-labeled wild-type or 19F-labeled (Y39F, Y125F, Y133F, Y136F) α-synuclein were performed as previously described [44, 49, 50].
Thioflavin T (ThT), TbCl3•6H2O, DyCl3•6H2O and LuCl3•6H2O from Sigma or Alfa Aesar were used without further purification. All other chemicals were of analytical grade from Sinopharm Chemical Reagent Co. Ltd.
Circular dichroism (CD) measurements
Samples containing 20 μM αS with different concentration of lanthanide metal ions in 10 mM MES and 100 mM NaCl at pH 6.0 were used for CD measurements. CD spectra were recorded on a ChirascanTM CD spectrometer in 10 mm cells from 260-190 nm with a step size of 1 nm and a bandwidth of 1 nm.
Samples comprised of 100 μM αS with 400 μM different metal ions in 10 mM MES, 100 mM NaCl, 500 μM phenylmethanesulfonyl (pH 6.0) were used for fibrillation experiments. αS fibril formation was induced at 37 °C by agitation with 220 rpm in the shaker. During fibrillation, small aliquots (10 μl) were removed and mixed with 1 mL 25 μM ThT. The fluorescence intensities were measured to monitor αS fibrillation. Fluorescence emission spectra were recorded at 482 nm on a HORIBA Fluoromax-4 spectrometer.
NMR Spectroscopy and Data Analysis
Availability of supporting data
All the supporting data are included as Additional file 1.
Nuclear magnetic resonance
Heteronuclear single quantum correlation
- Ln3+ :
Lanthanide metal ions
2-(N-morpholino) ethanesulfonic acid
- Rh :
This work is supported by Ministry of Science and Technology of China grant 2013CB910200 (C.L.), the 1000 Young Talents Program (C.L,), National Natural Sciences Foundation of China grant 21173258 (C.L.) and 21221064 (M.L.).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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