Journal of Molecular Biology
Volume 372, Issue 3, 21 September 2007, Pages 689-707
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Residual Structure, Backbone Dynamics, and Interactions within the Synuclein Family

https://doi.org/10.1016/j.jmb.2007.07.008Get rights and content

Abstract

The human synuclein protein family includes α-synuclein, which has been linked to both familial and sporadic Parkinson's disease, and the highly homologous β and γ-synuclein. Mutations in α-synuclein cause autosomal dominant early onset Parkinson's, and the protein is found deposited in a fibrillar form in hereditary and idiopathic forms of the disease. No genetic link between β and γ-synuclein, and any neurodegenerative disease has been established, and it is generally considered that these proteins are not highly pathogenic. In addition, β and γ-synuclein are reported to aggregate less readily than α-synuclein in vitro. Indeed, β-synuclein has been reported to protect against α-synuclein aggregation in vitro, as well as α-synuclein-mediated toxicity in vivo. Earlier, we compared the structural properties of the highly helical states adopted by all three synucleins in association with detergent micelles in an attempt to delineate the basis for functional differences between the three proteins. Here, we report a comparison of the structural and dynamic properties of the free states of all three proteins in order to shed light on differences that may help to explain their different propensities to aggregate, which in turn may underlie their differing contributions to the etiology of Parkinson's disease. We find that γ-synuclein closely resembles α-synuclein in its free-state residual secondary structure, consistent with the more similar propensities of the two proteins to aggregate in vitro. β-Synuclein, however, differs significantly from α-synuclein, exhibiting a lower predisposition towards helical structure in the second half of its lipid-binding domain, and a higher preference for extended structures in its C-terminal tail. Both β and γ-synuclein show less extensive transient long-range structure than that observed in α-synuclein. These results raise questions regarding the role of secondary structure propensities and transient long-range contacts in directing synuclein aggregation reactions.

Introduction

Synucleins are small, highly conserved vertebrate proteins that are highly expressed in neurons.1., 2., 3. In humans, three proteins, α-synuclein (aS), β-synuclein (bS), and γ-synuclein (gS), are included in the synuclein family.4., 5., 6. aS and bS are localized predominantly at presynaptic nerve terminals and are largely absent from peripheral tissues.7., 8. In contrast, gS is abundant in the peripheral nervous system and is expressed in other tissues, including brain, as well as breast and ovarian cancers.3., 6., 9., 10. In addition to their overlapping expression patterns, synuclein family members share a high level of sequence homology. The N-terminal domains of all three proteins, defined by their lipid-interactions, are especially highly conserved and include several imperfect 11 residue repeats, while the C-terminal domains are highly acidic and are more diverse between synucleins (Figure 1). Within the N-terminal domain, the most notable sequence difference between the proteins is the deletion of 11 residues in the bS sequence that correspond to parts of repeats 6 and 7 of aS and gS, and are located in the most hydrophobic region of the proteins, which is referred to as the NAC region in aS.11 Within the C-terminal domain, both aS and bS contain two 16 residue imperfect repeats,12 while gS has a relatively shorter C-terminal tail that does not contain these repeats. All three synucleins are intrinsically unstructured when isolated under physiological conditions,13., 14., 15., 16. and adopt highly helical structures in their N-terminal domains upon binding to lipid vesicles or detergent micelles in vitro.17., 18., 19., 20.

Among the three synucleins, aS has been intensively studied because it is linked with the etiology of Parkinson's disease (PD). aS is a major component of Lewy bodies and Lewy neurites,21 proteinaceous deposits associated with functionally damaged brain regions in PD, where it is found aggregated in the form of amyloid fibrils.22., 23. Furthermore, three missense mutations, A53T,24 A30P,25 and E46K26 in the α-synuclein gene are associated with familial Parkinsonism, and gene duplication27 or triplication28 can cause early onset familial PD. Despite extensive study, however, both the normal functions of aS and their relation to the mechanism by which aS contributes to PD remain unclear. Consistent with its localization to synaptic terminals, aS appears to be involved in synaptic plasticity.29 Additional and mounting evidence suggests a role for the protein in neurotransmitter release pathways. For example, aS has been shown to regulate the production of phosphatidic acid by phospholipase D,30 an activity that is thought to be important in the formation and fusion of synaptic vesicles.31 aS can act to rescue neuroegeneration caused by the absence of cysteine-string protein α, a protein responsible for assisting in fusion-mediating SNARE complex assembly.32 Accumulation of aS causes defects in endoplasmic reticulum-Golgi traffic in yeast, and increased neuronal expression of Rabs, proteins involved in vesicle trafficking, rescues aS-induced cell death.33 Finally, aS has been shown to modulate neurotransmitter release in a number of systems.34., 35., 36., 37., 38.

The role of aS aggregation in PD is unclear. All three PD-linked aS mutations increase the rate at which the protein forms oligomeric species in vitro,39., 40., 41., 42., 43. suggesting that this effect may underlie the ability of these mutations to cause disease. A number of models for how aggregation may lead to toxicity exist, but conclusive evidence for any one model has proven difficult to obtain. Nevertheless, extensive efforts have been made to understand the process by which aS converts into amyloid fibrils, including detailed structural studies of the free protein isolated in vitro,15., 44., 45., 46. representing the initiation point of the aggregation reaction, as well as of the aggregated fibrillar form of the protein, representing the end-point of the reaction.47., 48.

Unlike aS, relatively little is known about bS or gS. In contrast to aS, no mutation in the bS or gS genes in familial PD have been identified,49., 50., 51. and they are not found in Lewy bodies or Lewy neurites,23 excluding a primary role for either protein in the pathogenesis of PD. Nevertheless, some evidence exists for bS and gS deposition in PD or dementia with Lewy bodies,52 and for a genetic linkage between bS and dementia with Lewy bodies.53 In vitro, bS and gS are less prone to aggregation than aS. gS can form amyloid fibrils under conditions that lead to aS fibril formation, but does so less readily.14., 54. bS was initially reported to be incapable of forming fibrils,14., 54. but was shown recently to do so in the presence of metals.55 Interestingly, bS14., 56., 57. and gS14 were shown to inhibit aS fibril formation when incubated with aS in vitro. Additionally, double-transgenic mice, expressing human aS and bS show an ameliorated PD-like phenotype when compared with singly transgenic mice expressing human aS alone.56

Here, we report a detailed comparison of the structural properties of bS and gS with those of aS in the free state of each protein isolated in solution. Because bS and gS are not clearly linked to PD, despite their high level of sequence homology to aS, and are less prone to aggregate in vitro, structural differences between the three proteins may be informative regarding the properties of aS that are crucial for facilitating its aggregation, and for determining its role in the etiology of PD. We investigate the potential interactions of bS and gS with aS in an attempt to understand the inhibitory effects of the two proteins on aS aggregation and toxicity.

Section snippets

Circular dichroism (CD)

To assess the gross secondary structure content of all three synucleins under the conditions used in our NMR experiments, we collected far-UV CD spectra of all three proteins (Supplementary Data Figure 1). The spectra confirm the highly unstructured nature of the polypeptides, as indicated by the intense negative band near 195 nm and the absence of a strong signal at either the band around 218 nm characteristic of β-sheet or the bands at 208 and 222 nm indicating a helix . Nevertheless, the

The role of synuclein function and aggregation in disease

Despite the high degree of sequence homology between the members of the human synuclein family, only aS is clearly linked to the pathogenesis of PD. Within the N-terminal lipid-binding domain, extending through aS residue 94, bS contains 14 single residue substitutions and an 11 residue deletion when compared with aS, and gS contains 30 single residue substitutions. Within their C-terminal tails, a much greater degree of sequence diversity is evident for the synucleins, with 30 substitutions

Conclusions

Our results reveal a number of differences between the free states of aS, bS and gS. In particular, residual secondary structure in bS is strongly affected by the 11 residue deletion in the NAC region of this family member, resulting in the truncation of residual helical structure at position 65, the location where the highly helical structure of the micelle-bound state of bS becomes destabilized. In contrast, for both aS and gS, residual helical structure persists through the N-terminal

Materials

Recombinant aS, bS and gS were expressed in Escherichia coli BL21 (DE3) using plasmid constructs kindly provided by Dr Peter Lansbury (Department of Neurology, Harvard Medical School). To produce isotopically labeled proteins for NMR studies, saturated overnight LB-kanamycin cultures were used to inoculate M9 minimal media made with [U-13C]glucose and/or [15N]ammonium chloride. Cultures were grown at 37 °C to an A600 nm of 0.5–0.6, at which point protein expression was induced with 1 mM IPTG.

Acknowledgements

This work was supported by NIH/NIA grant AG019391, the Irma T. Hirschl Foundation, and a gift from Herbert and Ann Siegel (to D.E.). We thank Dr Peter Lansbury (Harvard Medical School) for the kind gift of expression vectors and Trudy Ramlall and Carla Rospigliosi for technical assistance. D.E. is a member of the New York Structural Biology Center, supported by NIH grant GM66354.

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