Perturbation in Long-Range Contacts Modulates the Kinetics of

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Perturbation in long-range contacts modulates kinetics of amyloid formation in #-Synuclein familial mutants Priyatosh Ranjan, and Ashutosh Kumar ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00149 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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ACS Chemical Neuroscience

Perturbation in long-range contacts modulates kinetics of amyloid formation in α-Synuclein familial mutants Priyatosh Ranjan1 and Ashutosh Kumar1* 1

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India ABSTRACT: The characteristic cross-β-sheet rich amyloid fibril formation by intrinsically disordered αSynuclein protein is one of the pathological hallmarks of Parkinson’s disease. Although unstructured in solution, presence of autoinhibitory long-range contacts in monomeric form prevents protein aggregation. Out of the various factors that affect the rate of amyloid formation, familial mutations play an important role in α-Synuclein aggregation. Eventhough these mutations are believed to form aggregation prone intermediate by perturbing these contacts, the correlation between perturbation and rate of fibril formation is not very straightforward. A combination of solution and solid-state NMR in conjunction with other biophysical methods has been used to identify the underlying mechanism behind the anomaly in the rate of aggregation for the novel mutants H50Q (fast aggregating) and G51D (slow aggregating). Perturbation of long-range contacts at the mutation sites and C-terminus in all the six familial mutants of α-Synuclein during the diseased condition (acidic pH) was observed. These contacts get rearranged at physiological pH resulting in the shielding of mutation sites. Additional contacts at the mutation site in slow aggregating mutant could be the reason for slower aggregation. Indeed, these contacts provide more rigidity to the monomeric G51D. Nonetheless, these mutations did not alter the overall secondary structure. The differential pattern of the long-range contacts at the monomeric level resulted in the perturbation of fibrillar core region as it became evident in the solid-state NMR spectra. Our results provide valuable insights in understanding the effect of long-range contacts on the aggregation of α-Synuclein and its mutants.

KEYWORDS: Parkinson’s disease, Lewy bodies, α-Synuclein, Long-range contacts, Chemical shift perturbation, Magic angle spinning.

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INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder that mainly affects the motor system.1 The disease is characterized by the loss of neuromelanincontaining dopaminergic neurons and the presence of intracellular inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs) in the substantia nigra pars compacta region of the midbrain.2,

3

LB consists mainly of proteinaceous aggregates of natively unstructured 140

residues presynaptic amyloidogenic protein α-Synuclein (α-Syn).4 Similar to LBs, LNs correspond to abnormal neurites that contain α-Syn filaments along with other proteins.5 The amino acid sequence of α-Syn is divided into three distinct domains. The highly conserved Nterminal (residues 1-60) region is dominated by the presence of imperfect tandem repeats with consensus core sequence K(A)-T(A,V)-K(V)-E(Q,T)-G(Q)-V(A), similar to the motifs that are found in the apolipoproteins.6 Although unstructured in solution, the residues 3-37 and 45-92 have been reported to form a pair of antiparallel curved α-helix in the presence of membrane.7 The residues 61-95 constitute a highly hydrophobic region essential for protein aggregation.8 This 35 amino acid residue peptide was first found in the SDS-insoluble fraction from the brain tissue of patients with Alzheimer’s disease (AD) and was named as non-Aβ component (NAC) of AD.9 A 12 amino acid stretch (V71-V82) of this hydrophobic domain is essential and sufficient for its fibrillization.10 The NAC region residues form βstrands in the amyloid fibrillar structure.11, 12 The C-terminal region (residues 96-140) is rich in negatively charged amino acids with five aspartatic acids and ten glutamic acids. The absence of secondary structure in this region might be due to the presence of five prolines which are known to induce turns and disrupt secondary structure.13 Despite having little or no ordered structure under physiological conditions, α-Syn exists as an ensemble of conformations that are stabilized by long-range contacts between N- and Cand NAC and C-terminus residues.14-16 These interactions shield the highly hydrophobic amyloidogenic NAC region resulting in the inhibition of amyloid fibril formation.16,

17

Recently, similar to the previous studies Esteban-Martin et al. used paramagnetic relaxation enhancement to identify fibrillar-like contacts in soluble monomeric α-Syn between residues forming β1-β2 and β4-β5 strands of the amyloid fibril. They also observed nonfibrillar contacts between residues present in β1-β3, β1-β4, β2-β4, and β2-β5 strands of the amyloid fibril.18 The release or redistribution of the long-range contacts is an essential factor for exposing the NAC region that enhances the aggregation and results into the amyloid fibril formation.15, 18 Even though the disordered monomeric form of α-Syn is the widely accepted structural model, other groups have shown that α-Syn can exist as an aggregation resistant


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tetramer.19,

20

However, this tetramer model was later questioned by several groups where

they reported monomeric and disordered nature of the protein inside the bacterial

21

and

22

mammalian cells. Conditions such as change in pH, temperature, and single point mutation perturb these long-range interactions leading to the formation of aggregation-prone partially folded intermediate

23

. The membrane-bound form of monomeric α-Syn protein adopts a

significant level of α-helical structure.7, 24-26 The structure of α-Syn bound to sodium dodecyl sulphate (SDS) micelle was determined by solution-state NMR and the formation of two curved α-helices (Val3-Val37 and Lys45-Thr92) connected by a well-ordered, extended linker was reported

7

(PDB ID: 1XQ8). Similarly, the structure of α-Syn protein in the

presence of binding partners like calmodulin (PDB ID: 2M55)27 and β-wrap proteins (4BXL)28 have been reported by several other groups. Till date, six PD-associated familial point mutations have been identified at the N-terminus of the protein: A53T,29 A30P,30 E46K,31 H50Q,32 G51D,33 and A53E.34 Even though previous studies suggests that these point mutations could perturb the native, autoinhibitory, longrange tertiary contacts resulting in the formation of aggregation-prone intermediate,15, 23 the correlation between the structural perturbation and fibrillization is complicated and remains to be established. While E46K,35 H50Q,36 and A53T 37 mutations enhance the rate of amyloid fibril formation, the contrary occurs in the case of A30P,38 G51D,39 and A53E

40

mutants.

Although significant difference in the lag phase has been observed for these mutants during aggregation, nothing noteworthy has been reported regarding the rationale behind the delay in aggregation at the residue specific monomeric level. Here, we aim to understand the mechanism behind the difference in the rate of fibril formation in familial mutants of α-Syn, i.e. H50Q and G51D using various biophysical experiments. However, for comparison, A30P, E46K, A53T, and A53E have been studied wherever required. The release or the redistribution of the long-range contacts in case of these mutants was studied using solution-state nuclear magnetic resonance (NMR) spectroscopy. Additionally, the effect of these point mutations on the residue-specific structural and dynamic properties of the monomeric protein was studied. The rate of the amyloid formation in the mutants was studied using thioflavin T (ThT) fluorescence and circular dichroism (CD) spectroscopy. The morphology of the formed fibrils and perturbations in the fibrillar spectra was studied using atomic force microscopy (AFM) and solid-state NMR respectively. Our results suggest that all the six familial mutants of α-Syn perturb the long-range interactions between the mutation sites and C- terminus at acidic conditions. However, due to the rearrangement of these contacts, no such effect was seen at



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the physiological pH with the exception of E46K. The presence of additional contacts involving lysine of the region 54-60 (K58 and K60) in G51D and A53E was speculated to be the reason for their slow aggregation. Even though the morphology of the fibrils formed by these mutants were similar to the WT as sen in transmission electron microscopy, significant perturbations were seen in the magic angle spinning (MAS) solid-state NMR spectra of fibrillar states.


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RESULTS AND DISCUSSION Perturbation of long-range contacts in the familial mutants of α-Syn To understand the effect of these point mutations on the conformation of the monomeric protein, 1H-15N HSQC spectra were recorded for all the synucleins (α-Syn WT and PD associated familial mutants). Well resolved sharp amide cross-peaks with a narrow dispersion (~ 0.8 ppm) in the HN dimension suggests the unfolded nature of the monomeric protein WT (Figure S1). The perturbation of the amide cross-peaks in the spectra of mutants were compared with the spectrum of WT. Figure 1A represents the amide region of the overlaid 1

H-15N HSQC spectra of WT (shown in red) and H50Q (shown in blue).

Figure 1. Residues showing significant chemical shift perturbations in H50Q and G51D. (A) Expansion of the overlaid 1H-15N HSQC spectra of WT (red) and H50Q (blue); and (B) WT (red) and G51D (green) recorded at pH 6.0. Residue specific chemical shift perturbation calculated for H50Q (C) and G51D (D). In G51D, the perturbation was extended at the mutation site (T54 - K60). Red, blue and green colour in figure C and D denote the basic Nterminal domain, the amyloidogenic NAC domain, and the acidic C-terminal domain respectively. Compared to the WT, significant chemical shift perturbations (CSPs) were observed for the residues (T44-A53) near the mutation site as well as for the residues located at the Cterminus in H50Q (D121 - Y133) (Figure 1C). The perturbation at the C-terminus suggests that these residues are in close contact with the mutation site, confirming the presence of



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long-range contacts as reported earlier.14, 16 Similarly, in case of the other mutant i.e. G51D (Figure 1B), significant perturbations were seen for the residues adjacent to the mutation site (V48 - T60) as well as for the residues at the C- terminus (D121 - Q134) (Figure 1D). Interestingly, compared to H50Q, the perturbation was extended at the mutation site in the case of G51D involving residues T54-K60. However, additional perturbation of residues 44 and 45 in H50Q suggests the proximity of these residues at the mutation site. α-Syn amyloid formation is dictated by the nature of amino acid at a particular position. While A53T aggregates faster,37 the other mutant A53E retards the rate of amyloid fibril formation.40 To understand the effect of these point mutations on the long-range interactions, CSPs were calculated for the familial mutants A53T and A53E. Figure S1C and D represents the overlaid 1H-15N HSQC spectra of WT with A53T and A53E. Similar to the other familial mutants, these mutations did not affect the amide proton dispersion range (~0.8 ppm) suggesting that the monomeric protein remains unfolded in nature. In the case of A53T, significant CSPs were observed for residues H50-A56 and D121-Y133 (Figure 2A).

Figure 2. Significant CSPs were observed at the mutation site as well as for the residues at the C-terminus in familial mutants of α-Syn at pH 6.0. Residue-specific chemical shift perturbation calculated for A53T (A), A53E (B), A30P (C), and E46K (D). Significant CSPs were seen only at the mutation site in H50Q (E) and G51D (F) at pH 7.4. Red, blue and green colour denotes the basic N-terminal domain, the amyloidogenic NAC domain, and the acidic C-terminal domain respectively. In additions to these residues, CSPs were also seen for residues E57-K60, in A53E mutant, similar to G51D (Figure 2B). The effect of point-mutation was also studied for other familial mutants, i.e., A30P and E46K (Figure S1E and F). In both the mutants, significant CSP was


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seen at the mutation site (G25-K34 in A30P and V40-H50 in E46K) as well as for the residues at the C-terminus (Figure 2C and D) suggesting the perturbation of long-range contacts in these mutants as well. The perturbation of His50 in A30P mutant indicated spatial closeness of residues near Pro30 and His50. The above studies suggest that C-terminus residues (mainly D121- D135) are in close contact with the mutation sites; located at the N-terminus (all the six disease-associated mutations are located at the N-terminus). Perturbation of long-range contacts in all these familial mutants results in the formation of destabilized conformations with the exposure of highly hydrophobic amyloidogenic region. Even though the perturbations should accelerate the fibril formation, the case is not true for all the familial mutants. While H50Q and A53T aggregate faster,36, 37 the rate of fibril formation gets retarded in G51D and A53E.39, 40 This is due to the fact that presence of additional electrostatic interactions in case of G51D and A53E, which could be delaying the exposure of hydrophobic region exist between the negatively charged amino acids i.e. aspartic and glutamic acid with the lysines of the region 54-60 (K58 and K60). This can be noticed by significant CSP where extended perturbation was observed involving residues T54-K60 both in the case of G51D and A53E. Thus, it can be said that all these familial mutants destabilize the long-range interactions resulting in the formation of partially folded intermediate. Although the intermediates are aggregation-prone in A53T and H50Q, the same is not true for the intermediates generated in the case of G51D and A53E. For the familial mutants A30P and E46K, apart from C-terminus to N-terminus contacts, other physicochemical properties such as net charge and secondary structure propensity play an important role in determining the rate of amyloid fibril formation as reported previously.41 Rearrangement of long-range contacts with a change in pH Metabolic acidosis is a common phenomenon that is observed in the brain of the patients suffering from various neurological disorders including PD.42, 43 Therefore, the experiments were performed at slightly acidic pH. This would also help us to observe the exchangeable amide protons which are not possible at higher pH. However, in order to observe the effect of pH on the long-range interactions, CSPs were also calculated at physiological pH (pH 7.4) for the familial mutants H50Q and G51D. Figure S2A represents the overlaid 1H-15N HSQC spectra of H50Q and G51D at pH 7.4. In both the mutants, CSPs were seen only at the mutation sites and no such effects were observed either at the N- and C-terminus (Figure 2E and F). Similar to pH 6.0, the perturbation was extended at the mutation site in G51D (residues H50-K60) compared to H50Q (V48-G51).



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Thus at physiological pH, the mutation sites are no longer in contact with the residues at the N- or C-terminus and no significant perturbations are observed beyond the mutation site. Further, we also studied the redistribution of long-range contacts at two different pH for the WT and the familial mutants H50Q and G51D (Figure 3 and Figure S2B).

Figure 3. Residues showing significant perturbations at different pH in the overlaid 1H15 N HSQC spectra. Expansion of the overlaid 1H-15N HSQC spectra recorded at two different pH (pH 6.0 and pH 7.4) for WT (A), H50Q (B) and G51D (C). In all the synucleins, the spectra recorded at pH 6.0 has been shown in red while the spectra recorded at pH 7.4 has been shown in green, blue, and black for WT, H50Q, and G51D respectively. With a change in pH from 6.0 to 7.4, significant perturbations were seen for the residues V3K6 (N-terminus), A124-M127 and Y133-D135 (C-terminus) (Figure 4A) in WT and its mutants. This might be due to the presence of overall net positive and negative charge at the N- and C-terminus respectively. The rearrangement of long-range contacts at physiological pH resulted in the close association of initial N- and C-terminus region resulting in the shielding of highly hydrophobic amyloid forming region. In case of the WT and G51D,


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perturbation was also observed for residue H50. This suggests the proximity of histidine either with the N- or C-terminus or the change in the protonation state of imidazole ring at two pH values (Figure 4A).

Figure 4. Redistribution of long-range contacts and residue specific secondary structure in α-Syn familial mutants. (A) Residue-specific CSP due to change in pH was calculated for WT (top panel), H50Q (middle panel) and G51D (bottom panel). In all the synucleins, significant CSPs were observed for the residues at the N- and C-terminus. (B) Hα secondary chemical shifts calculated for monomeric α-Syn WT (top panel), H50Q (middle panel) and G51D (bottom panel). Red, blue and green colour denotes the basic N-terminal domain, the amyloidogenic NAC domain, and the acidic C-terminal domain respectively. The perturbations of the chemical shift due to the other four point mutations (A30P, E46K, A53T, and A53E) were also studied at physiological pH. Compared to pH 6.0 CSP calculation showed perturbation only at the mutation site in case of A30P, A53T and A53E (Figure S3). However in case of E46K, significant CSP was also seen for the C-terminus residues (D121-E126 and Y133-E137) indicating that these residues are in long-range contacts with the mutation site, unlike other mutants at pH 7.4. Next, we also studied the redistribution of long-range contacts in these mutants. 1H-15N HSQC spectra were recorded for these mutants at pH 6.0 and pH 7.4 and CSPs were calculated (Figure S4 and Figure S5). In all the mutants, with a change in pH (from pH 6.0 to 7.4), significant perturbations were seen for the residues V3-K6 (N-terminus), E123-E130 and Y133-E137 (C-terminus) indicating the close association of initial N- and C-terminus region, similar to H50Q and G51D. The increased CSP for H50 in all these cases can be explained on the basis of either its proximity with the N- or C-terminus or change in the protonation state of histidine as explained above.



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From these studies, it can be summarized that α-Syn and its mutants behave differently at different pH. While at pH 6.0, mutation sites were in contact with the C-terminus residues (D121-Q134) through long-range interactions; these contacts were missing at pH 7.4. Instead, at pH 7.4, long-range interactions were seen at N- (V3-K6) and C-terminus (~A124-M127 and ~Y133-D135). Due to the redistribution of long-range contacts at physiological pH, Cterminus residues were no longer in contact with the mutation sites. At both the pH, additional contacts were seen with the region V55-K60 in the case of G51D and A53E. This auto-inhibitory electrostatic interaction could be the reason for the slower aggregation of G51D and A53E as compared to the other mutants. Residue-specific secondary structure and dynamics of α-Synuclein WT and its mutants H50Q and G51D Deviations of observed chemical shifts from the random coil values (secondary chemical shift), especially for

13 α

C,

13 β

C , and 1Hα provide a convenient and sensitive probe of

secondary structural propensities.44 The random coil chemical shifts data for the 20 common amino acids in 8M urea at pH 2.3 were used from the previous studies.45 The sequence correction of random coil chemical shifts was done using neighbour correction factors as previously reported.46 In α-helices, 13Cα and 13CO resonances are shifted downfield (positive) while

13

Cβ and 1Hα resonances are shifted upfield (negative) from their positions in the

random coil states.47 The opposite pattern is seen in case of the β-sheets. The difference between the observed and random coil chemical shifts in disordered/unfolded proteins is much smaller compared to the folded proteins.45 1Hα secondary shifts were used to study the residue specific secondary structure of the monomeric protein WT and its mutants (Figure 4B). Secondary chemical shift values reported here are the differences between the measured Hα chemical shift and the sequence corrected random coil value for the corresponding amino acid.48 Compared to the WT, no such significant effect on the residue-specific secondary structure of the protein was observed in the mutants suggesting that these mutations have no global effects on the structural propensities of the protein (Figure S6A and B). Though the shifts are small, the pattern of the deviations in WT and its mutants shows a negative bias suggesting that these residues have a significant preference for helical secondary structure. Positive Hα values for residues 37-40 in WT indicated that these residues are prone to form βstrand. Residues 37-40 are a part of first β-strand in the α-Syn fibrillar structure as reported previously.11, 12 In all the synucleins, the C-terminus showed a random pattern suggesting it is highly disordered part of the protein. Hence, the familial mutants did not affect the overall


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secondary structure of the protein and it remains disordered in nature with a tendency to form helix.

Measurement of residue-specific backbone dynamics To study the effect of point mutation on the backbone dynamics, we measured 15N transverse relaxation rate (R2) and steady state

15

N-{1H} heteronuclear NOE (Figure 5). Both the

parameters report on fast (nanosecond to picosecond) motions of the NH bond vector, while R2 values are also sensitive to microsecond-millisecond (µs-ms) time-scale conformational or chemical exchange.49 Typical values of the 15N-{1H} NOE and R2 parameters for residues in the core of well structured proteins are around 0.8 and 10 s-1.50 Lower values of the 15N-{1H} NOE parameter are indicative of a greater degree of fast motions, whereas higher values of R2 indicate a greater degree of slower motions. For α-Syn WT, average R2 values of ~4.6 s-1 and average

15

N-{1H} NOE values near 0.2 was calculated. The average R2 and

15

N-{1H}

NOE values for the WT and the mutants H50Q and G51D in a region wise manner have been shown in Table 1. Compared to WT, an increase in the flexibility for the residues at the mutation site (V48-G51) was seen in case of H50Q (Figure S6C).

Figure 5. Residue-specific backbone dynamics calculated for α-Syn wild-type and its mutants. (A) 15N transverse relaxation rates (R2) for α-Syn WT (top panel), H50Q (middle panel), and G51D (bottom panel). (B) 15N-{1H} heteronuclear NOE calculated for WT (top panel), H50Q (middle panel), and G51D (bottom panel). Red, blue and green colour denotes the basic N-terminal domain, the amyloidogenic NAC domain, and the acidic C-terminal domain respectively.



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However, no such change was seen in the mutant G51D (Figure S6D). In both the familial mutants, a decrease in the mobility (increased rigidity) was observed for the stretch of residues from G41-T44. Overall, G51D showed higher average R2 value for the three domains compared to the WT and H50Q (Table 1). Taken together dynamics measurements suggested that the monomeric G51D is comparatively rigid in nature. This is also supported by the fact that the presence of additional contacts involving residues T54-K60 in case of G51D makes it more rigid, unlike WT and H50Q. The residue-specific backbone dynamics was also calculated for the other four PD-associated mutants (A30P, E46K, A53T, and A53E) and compared with α-Syn WT. The relaxation parameters observed for all four α-Syn mutants indicate a much greater mobility than that found in well structured proteins indicating that all four mutants are predominantly unfolded (Figure S7). The average R2 and

15

N-{1H} NOE values for the WT and the mutants in a

region wise have been shown in Table S1. R2 and 15N-{1H} NOE data for the mutants A30P, A53T and A53E showed values similar to the WT and no such significant difference was observed (Figure S8). However, compared to the WT, an overall increase in the slower motions was seen for N-terminus residues in case of A53T. Higher value of average R2 for the N- (V37-S42) and C-terminus (residues E123-E139) in case of E46K suggested that the monomeric form is more rigid compared to the WT. Lower values of R2 for the NAC region in case of E46K and A53E indicated that this region is more flexible compared to the WT. Interestingly, V66 (in case of A53E) and V70 (both in case of E46K and A53E) showed positive heteronuclear NOE value compared to the WT and other mutants. Overall, it can be said that the familial mutation E46K has a significant effect on the dynamics of the monomeric form making the N- and C-terminus more rigid as reported previously.51


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Table 1. Average R2 value calculated for the individual domain in α-Syn WT, H50Q, and G51D. Synuclein and its familial mutants

R2 (sec-1)

15

N-{1H} NOE

WT (1-60)

4.99 ± 0.18

0.26 ± 0.04

WT (61-95)

4.64 ± 0.17

0.19 ± 0.04

WT (96-140)

4.16 ± 0.11

0.21 ± 0.04

H50Q (1-60)

5.15 ± 0.25

0.29 ± 0.05

H50Q (61-95)

4.74 ± 0.26

0.22 ± 0.05

H50Q (96-140)

4.03 ± 0.14

0.22 ± 0.05

G51D (1-60)

5.48 ± 0.28

0.28 ± 0.03

G51D (61-95)

5.00 ± 0.30

0.19 ± 0.03

G51D (96-140)

4.27 ± 0.16

0.21 ± 0.03

Aggregation kinetics of α-Synuclein and its mutants α-Syn follows a classical nucleation-dependent polymerization reaction in which the soluble monomeric state undergoes a conformational transition to form insoluble amyloid fibrils.52 ThT is a fluorescent dye that binds to the cross-β-sheet rich amyloid fibrils and exhibits an increase in the fluorescence emission (~ 480 nm). Hence, this assay was used to monitor the fibrillation process.53 For aggregation experiments, LMW α- Syn and its mutants were purified and adjusted at a concentration of 200 µM in 20 mM phosphate buffer, (pH 6.0 as well as pH 7.4) containing 0.01% sodium azide. The aggregation reaction was initiated by incubation of the samples at 37 °C with slight agitation. The kinetics determined by ThT fluorescence suggested the formation of amyloid fibrils through nucleation-dependent polymerization reaction in all the synucleins (Figure 6A and B). Similar to our previous studies and by other research groups, the aggregation kinetics of H50Q was faster compared to the WT, while the rate of amyloid fibril formation was slower in G51D.36, 39



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Figure 6. α-Syn aggregation monitored at two different pH using ThT fluorescence, CD and AFM. (A) The aggregation rate of H50Q was faster compared to the WT while the retardation of amyloid fibril formation was seen in the mutant G51D at pH 6.0. (B)At physiological pH, the pattern of aggregation was similar to that of pH 6.0. However, an overall decrease in the rate of the fibril formation was seen for the samples. Secondarystructural changes during the course of fibrillation was monitored using CD spectroscopy at pH 6.0 (C) and pH 7.4 (D). The morphology of the formed fibrils during the course of aggregation was monitored using AFM at pH 6.0 (E) and pH 7.4 (F) at the end of stationary phase. Scale bars are 500 nm. Secondary-structural changes during the course of fibrillation were monitored with the help of CD spectroscopy. A characteristic β-sheet formation in H50Q (~26 h) was seen at an early time point compared to the WT (~55 h) and G51D (~100 h) (Figure 6C). Since the rearrangement of long-range contacts was seen at physiological pH in NMR studies, the aggregation kinetics was also performed at pH 7.4 (Figure 6B). Although there was a significant decrease in the rate of fibril formation at pH 7.4, the pattern of aggregation was similar at both the pH. While H50Q took ~46 h for the formation of β-sheet, WT and G51D


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took ~69 h and ~150 h respectively (Figure 6D). A significant decrease in the rate of fibril formation at pH 7.4 might be due to the fact that NAC region is more shielded at physiological pH compared to pH 6.0 as concluded from NMR results. Hence, exposure of NAC region requires breaking these contacts and therefore statistically need more time for the amyloid fibril formation. The slower aggregation of G51D can be explained on the basis of additional contacts as seen in NMR studies at both the pH. The morphology of the formed fibrils, monitored using AFM at the end of aggregation showed highly-ordered long fibrillar networks for all the synucleins samples (Figure 6E and F). Effect of the familial mutants on the morphology and structure of the fibrils The amyloid fibrils are the end stage product of α-Syn aggregation. The morphology of the fibrils of the mutants H50Q and G51D was compared with the WT. Similar to AFM studies, no distinguishable changes in the fibril morphology of the mutants were observed by the transmission electron microscopy studies. All the samples displayed long fibrils with a diameter of ~ 10-13 nm (Figure 7A). Next, we compared the residue-specific chemical shift perturbations in the fibrillar structure using MAS solid-state NMR. For solid-state NMR analysis, uniformly doubly labelled [13C, 15

N] α-Syn WT, H50Q, and G51D fibrils were prepared by expressing the proteins in M9

minimal media, supplemented with 15N labelled NH4Cl and 13C U-labeled glucose as a sole source of nitrogen and carbon respectively. Figure 7B shows the comparison of twodimensional (2D) PDSD (proton-driven spin diffusion) spectra with a mixing time of 50 ms and spinning at 10 KHz recorded on α-Syn WT, H50Q, and G51D fibrils. PDSD is a standard experiment which provides initial information about the molecular organization and the quality of the fibril prepared for structural studies. All 13C atoms within a certain distance of one another are correlated by cross-peaks in the spectrum. At short mixing times (~ 50 ms), all carbon atoms within one spin-system (intra-residue) are coupled to one another whereas at longer mixing times (~150 ms), both intra- and inter-residue correlations appear. Solid-state NMR samples of α-Syn WT and the mutants yielded high resolution 13C-13C 2D spectra with narrow linewidths (~1-1.5 ppm) indicating a high degree of homogeneity throughout the fibrils. The characteristic amino acid regions in the 2D 13C-13C PDSD spectra of α-Syn WT were compared with the previously assigned spectra 11, 12, 54. From the spectral comparison, a difference between α-Syn WT fibrils and the fibrils of mutants (H50Q and G51D) are evident as highlighted with the straightforward I88 (Isoleucine 88) assignment (Figure 7B). The presence of the three carbon cross-peaks in the Cα (~54.2-57.9 ppm) and Cβ (63.9-67.8 ppm)



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region suggested the involvement of S9, S42 and S87 in the fibrillar core region (Figure S9) as reported previously.54 However, the presence of a single serine cross-peak (58.6-64.3ppm) in H50Q is indicative of the perturbation in the length of the fibrillar core. Similarly, the shifts of the serine cross-peaks in G51D suggested the fibrillar core perturbation. This was further corroborated with the shifts of the cross-peaks in the region corresponding to amino acids threonine, alanine, valine and isoleucine (Figure S9 and S10).

Figure 7. Fibrils of familial mutants H50Q and G51D are highly homogeneous and morphologically similar to WT fibrils. (A) Comparison of the electron micrographs of WT (top), H50Q (middle), and G51D (bottom). (B) Overlaid 13C-13C 2D PDSD spectra of WT (red) and H50Q (blue, top panel) and WT (red) and G51D (green, bottom panel). From the above studies, it is clear that both the mutants i.e. H50Q and G51D contain β-sheet elements, but their number, length and distribution may differ as can be discerned from the perturbed fibrillar spectra. The perturbed chemical shifts of Ile88 and other amino acids (Figure S4 and S5) are due to the disturbance of chemical environment by the mutation at position 50 (H50Q) and position 51 (G51D). A more detailed comparison between these fibrillar mutants requires the sequential resonance assignments of these fibrils which are in progress.


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CONCLUSION Here we present the molecular details of the underlying mechanism behind the differential rate of aggregation in α-Syn familial mutants. During diseased conditions (metabolic acidosis), perturbation of long-range contacts between the mutation sites and C-terminus of the mutants results in exposure of highly hydrophobic amyloidogenic region which leads to amyloid fibril formation. The rearrangement of these contacts at physiological pH leads to the shielding of fibrillar core region thereby retarding the rate of amyloid fibril formation. In case of the recently discovered familial mutants G51D and A53E, the presence of additional electrostatic interactions/contacts between negatively charged amino acid with lysines of the region T54-K60 is responsible for delaying the rate of fibril formation. An enhanced 15N R2 value for the monomeric G51D indicates the increased rigidity due to these contacts compared to WT and H50Q. However, CD and secondary chemical shift analysis have shown that these mutants do not have any significant secondary structure perturbation at the monomeric level. MAS solid-state NMR studies of these mutant fibrils suggest that perturbation of the fibrillar core region arises due to the differences in the pattern of longrange contacts in the monomeric structure. These studies will help us in the better understanding of the role of long-range contacts perturbation on the differential rate of α-Syn aggregation.



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METHODS Purification of Synucleins (α-Syn wild-type, A30P, E46K, H50Q, G51D, A53T and A53E) Unlabeled and isotope-labeled Homo sapiens α-Syn wild-type (WT) and its familial mutants A30P, E46K, H50Q, G51D, A53T and A53E were expressed in E.coli BL21 (DE3) strain and purified according to the established protocol.55 Bacterial cells were grown in LB media with ampicillin as a selectable marker. Uniformly

15

N labeled samples were prepared in M9

15

minimal media containing N NH4Cl as the sole source of nitrogen. The protein expression was induced for 4 hours after the addition of 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were then harvested by centrifugation. The cell pellet was dissolved in the lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail (Roche, Basel, Switzerland) to avoid any proteolytic cleavage. Cells were then sonicated using probe sonicator followed by heating at 95°C for 10-15 minutes. The supernatant was collected to a fresh tube and streptomycin sulphate (10%; 136µl/ml supernatant) and glacial acetic acid solutions (228µl/ml supernatant) were added, followed by centrifugation (14,000 rpm, 30 minutes, 4ºC). The resulting supernatant was precipitated by saturated ammonium sulphate prepared at 4°C. Precipitated protein was collected by centrifugation (14,000 rpm, 30 minutes, 4ºC) and the pellet was washed with 50% ammonium sulphate solution followed by centrifugation (14,000 rpm, 30 minutes, 4ºC). Washed pellet was resuspended in 100 mM ammonium acetate followed by adding an equal volume of ethanol. It was then centrifuged and the above step was repeated thrice followed by final resuspension in 100 mM ammonium acetate solution.

Preparation of low molecular weight (LMW) α-Synuclein For aggregation studies, α-Syn protein solution was dialyzed in Slide-A-Lyzer mini dialysis tubes of 10 kDa cutoff against 20 mM sodium phosphate buffer, pH 6.0 4°C. LMW form of α-Syn was isolated using amicon ultra 100 kDa cut-off filters (Millipore) according to previously described method.56 LMW was shown to contain mostly monomers along with some amount of low-order multimers. For NMR studies, the dialysed protein was passed through 30 kDa cut-off filter (Millipore) where LMW α-Syn contains mostly monomeric and/or dimeric protein. The pH of the resulting solution was confirmed using pH meter microelectrode (Cyberscan pH 2100, Eutech instruments, Singapore). Similar to pH 6.0, LMW α-Syn samples at pH 7.4 were also prepared.


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Thioflavin T (ThT) fluorescence assay For amyloid fibril formation, LMW α-Syn WT along with the familial mutants H50Q and G51D (~250 µM) was incubated at 37 ºC with slight agitation. The fibrillation kinetics was initiated in 20 mM phosphate buffer, (at pH 6.0 as well as pH 7.4) containing 0.01% sodium azide. The protein concentration was diluted to obtain final concentration of 10 µM and 2 µl of 1mM ThT solution was added to it. ThT fluorescence assay was monitored using JASCO spectrofluorometer FP-8500 with excitation at 450 nm and emission in the range of 460-500 nm. The excitation and emission slit width was kept at 5 nm. ThT fluorescence at 482 nm was plotted for all samples against incubation time. The experiment was repeated three times.

Circular dichroism spectroscopy (CD) measurement Secondary structural changes during the course of aggregation reaction were monitored using CD. For CD, 12 µl of 250 µM protein solution was diluted to 200 µl in 20 mM sodium phosphate buffer, to obtain a final protein concentration of 15 µM. The solution was transferred into 0.1 cm path-length quartz cell (Starna, Hainault, London). Spectra were acquired using JASCO- J-1500 CD spectrometer (Easton, Maryland) at 25°C over the wavelength of 198-260 nm. For signal averaging, three independent readings were taken. Raw data were processed by spectra smoothing and subtraction of buffer spectrum.

Atomic Force Microscopy (AFM) Morphology of the fibrils formed at the end of aggregation reaction (100 hours) were observed using AFM (Asylum research, Santa Barbara, CA). Small aliquots of the aggregation reactions were taken and diluted to a final concentration of 30 µM. Samples were then spotted on a freshly cleaved mica sheet, incubated for 5~10 minutes, washed with double distilled water and then dried at room temperature for 40 minutes. The imaging was performed in tapping mode with silicon nitride cantilever. The scan rate was kept at 1Hz and 2-3 different areas were randomly scanned.

Transmission Electron Microscopy (TEM) To examine the morphology of the amyloid protein fibrils under EM, aliquots of protein samples were diluted to obtain a final concentration of ~ 50 µM. The diluted solutions were then spotted on a glow-discharged, carbon-coated formvar grid (Electron microscopy Sciences, Fort Washington, PA) and incubated for 5-8 min. The grids were then washed with



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double distilled water, and stained with 1% (w/v) aqueous uranyl acetate solution. Uranyl acetate solution was freshly prepared and filtered through 0.2-micron sterile synringe filter (Millipore). The imaging of samples was done at 120 kV with nominal magnifications between x26,000, and x43,000. Images were recorded using Megaview imaging system.

Nuclear Magnetic Resonance (NMR) Study NMR data acquisition, processing and analysis NMR experiments were carried out on a Bruker Avance AV 800 MHz spectrometer equipped with 5mm HCN triple resonance cryo-probe with an actively shielded Z-gradient and Bruker Ascend 750 MHz equipped with room temperature 5 mm TXI probe. All the experiments were performed at 15°C in 20 mM sodium phosphate buffer (at pH 6.0 as well as pH 7.4) and (90:10) H2O/D2O ratio.

α-Synuclein sequence-specific resonance assignment α-Syn is a widely studied protein and the backbone (1H,

13

C, and

15

N) chemical shift

assignments for this disordered protein is submitted to biological magnetic resonance data bank (BMRB) by several research groups.57-59 In this study, the backbone resonance assignment for α-Syn protein was done using a combination of triple resonance experiments, mainly HNN and an efficient reduced dimensionality (RD) based method published previously.60 The assignment of the side chain hydrogen atoms was done through

15

N-

TOCSY-HSQC.61 For TOCSY-HSQC experiment, spectral widths comprised 14 ppm (1H, F1), 26 ppm (15N, F2) and 10 ppm (1H, F3) with isotropic mixing step of 75 milliseconds. Time domain data consisted of 128 (t1) x 48 (t2) x 2048 (t3) complex points. For the familial mutants of α-Syn, two-dimensional excitation sculpting WATERGATE 1H-15N heteronuclear single quantum correlation (HSQC) spectra was 14 ppm and that for

15

62, 63

were collected. The spectral width for 1H

N was 30 ppm. Free induction decays were collected with 2048

and 256 complex t2 and t1 points respectively. The amide cross-peaks which showed significant perturbations in the 1H-15N HSQC spectra were further confirmed with the help of 15

N-TOCSY-HSQC.

NMR data were processed with Topspin 3.2 version (BRUKER, http:// www.bruker.com) and analysed with Sparky 3.113 64 and CCPNmr.65


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Chemical Shift Perturbation (CSP) Perturbation of amide cross-peaks chemical shifts due to the familial mutants was calculated using the formulae:

(5∆ ) + (∆N )

(Where ∆δHN = difference in the amide proton cross-peak chemical shift; δ15N = difference in the

15

N chemical shift). The cutoff was decided according to the previously published

method.66

Secondary Chemical shift calculation Hα Secondary chemical shift is defined as:

∆ = ( ) − ( ) The sequence-corrected Hα secondary chemical shift (∆δ) was calculated to analyze the secondary-structure propensities of the disordered protein according to the previously published protocol.46 The random coil chemical shifts were taken from previous study 45 that uses 8M urea at pH 2.3, 20 ºC for measurements on peptides to arrive at the random coil chemical shifts. Residue-specific dynamics studies In order to study the effect of familial mutations on residue-specific dynamics, 15N transverse relaxation rates (R2) were measured using CPMG delays: 16.96, 33.92, 50.88*, 67.84, 84.81, 118.72, 136*, 153, and 187 ms (* indicates repeat points). Intensities of the peaks were measured using Topspin 3.2. T2 values with their errors were extracted from mathematica 5.2 ‘Relaxation Decay Analysis’ developed in Spyracopoulos’s lab.67 The error (∆R) in R2 was calculated from the data fitting errors as:

∆

where ∆T2 is the standard deviation in the T2 fitting. Steady-state 15N-{1H} heteronuclear NOE measurements were carried out with a total 5 sec interscan delay where proton saturation time was 3 s and relaxation delay was 2 s. For the experiment without proton saturation, the relaxation delay was 5 s. The NOEs were



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calculated as Isat./Inonsat.; where Isat. and Inonsat. are the peak intensities in the presence and absence of proton saturation. Errors were calculated using the following equation:

!"# = ($%&' /$)*)%&' ) +(!%&' /$%&' ) + (

!)*)%&' ) $)*)%&'

where σsat. and σnonsat. are the root-mean-square noise values for each of the peaks in presence and absence of proton saturation. Solid-state NMR data collection and analysis α-Synuclein fibril preparation for solid-state NMR experiments Solutions of monomeric α-Syn WT, H50Q, and G51D (20 mM sodium phosphate buffer, pH 7.4, 0.02% azide) were incubated at 37°C with slight agitation (50-100 rpm). After 3 weeks of fibrillation, samples were centrifuged at 30,000 rpm for 1 hour. The resultant pellets were washed with buffer and centrifuged again for 1 hour at 30,000 rpm. This step was repeated 34 times. The obtained pellet was then packed into 3.2 mm (outer diam. 3.2 mm, ext. length 15.4 mm, temperature range -30 to 70 °C, maximum speed 24 kHz) zirconia MAS rotor and stored at 4°C until further use. All solid-state NMR experiments were carried out on a 750 MHz (17.6 T) Bruker Ascend spectrometer (Bruker Biospin, Germany) using 3.2 mm E-free triple-resonance (1H, 13C, 15N) probe head operating at 10 kHz MAS with a sample temperature at 10°C. One-dimensional (1D) cross polarisation (CP) and two-dimensional (2D) Proton-Driven Spin Diffusion (PDSD), NCA and NCO spectrum of synucleins fibrils were acquired.

13

C-13C correlation

experiments were conducted using PDSD 68 with different mixing times (10, 50, and 150 ms). Spectral widths comprised 220 ppm (13C, F1) and 260 ppm (13C, F2) with t1max and t2max of ~7.7 ms and ~13.05 ms respectively. The chemical shifts were calibrated using DSS (sodium salt of 2, 2-dimethyl-2-silapentane-5-sulphonic acid) as an internal reference.69 NMR data were processed with Topspin 3.2 version (BRUKER, http:// www.bruker.com) and analysed with CCPNmr.65


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ABBREVIATIONS PD, Parkinson’s disease; LB(s), Lewy bodies; LN(s), Lewy neurites; SNpc, Substantia nigra pars compacta; α-Syn, α-Synuclein; NAC, Non-amyloid-β component; ThT, Thioflavin T; CD, Circular dichroism; AFM, Atomic force microscopy; TEM, Transmission electron microscopy; CSP, Chemical shift perturbation; R2, Transverse relaxation rate; NOE, Nuclear overhauser effect; MAS, Magic angle spinning; PDSD, Proton-driven spin diffusion.

AUTHOR INFORMATION Corresponding Author Phone: 91-22-2576-7762. E-mail: [email protected]

Author Contributions P.R. and A.K. designed the research. P.R. performed the experiments. P.R. and A.K. analyzed the data and wrote the manuscript.

Funding Sources This work was supported by Ramalingaswamy re-entry fellowship (BT/RLF/Reentry/22/2010) from Department of Biotechnology, Government of India to A.K. P.R. is grateful to MHRD (Government of India) for his fellowship.

ACKNOWLEDGEMENTS The authors acknowledge HF NMR, Bio-AFM and electron microscopy facility, funded by RIFC, IRCC, IIT Bombay. We thank Prof. Samir K. Maji (IIT Bombay) for α-Syn wild type and mutant clones. We are also very grateful to TIFR Mumbai for providing National facility for High-Field NMR and thankful to NMR research center, IISc Bangalore for 800 MHz NMR time. We thank Dr. Jithender G. Reddy for the help in setting up NMR experiments. We are also very thankful to Rajlaxmi Panigrahi for providing valuable inputs during the manuscript preparation.



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