A Structural and Functional Role for 11-mer Repeats in α-Synuclein and Other Exchangeable Lipid Binding Proteins

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Abstract

We have used NMR spectroscopy and limited proteolysis to characterize the structural properties of the Parkinson's disease-related protein α-synuclein in lipid and detergent micelle environments. We show that the lipid or micelle surface-bound portion of the molecule adopts a continuously helical structure with a single break. Modeling αS as an ideal α-helix reveals a hydrophobic surface that winds around the helix axis in a right-handed fashion. This feature is typical of 11-mer repeat containing sequences that adopt right-handed coiled coil conformations. In order to bind a flat or convex lipid surface, however, an unbroken helical αS structure would need to adopt an unusual, slightly unwound, α11/3 helix conformation (three complete turns per 11 residues). The break we observe in the αS helix may allow the protein to avoid this unusual conformation by adopting two shorter stretches of typical α-helical structure. However, a quantitative analysis suggests the possibility that the α11/3 conformation may in fact exist in lipid-bound αS. We discuss how structural features of helical 11-mer repeats could play a role in the reversible lipid binding function of α-synuclein and generalize this argument to include the 11-mer repeat-containing apolipoproteins, which also require the ability to release readily from lipid surfaces. A search of protein sequence databases confirms that synuclein-like 11-mer repeats are present in other proteins that bind lipids reversibly and predicts such a role for a number of hypothetical proteins of unknown function.

Introduction

α-Synuclein (αS) is a synaptic vesicle-associated protein that aggregates into amyloid fibrils that comprise a major component of the Lewy body deposits found primarily in the substantia nigra of Parkinson's disease (PD) victims.1 αS is genetically linked to PD through two autosomal dominant mutations, A30P and A53T,2., 3. which lead to early onset disease, and which cause a corresponding increase in the oligomerization rate of the protein in vitro.4., 5., 6. Transgenic flies and mice expressing wild-type or mutant αS exhibit PD phenotypes, including death of dopaminergic neurons (in flies), formation of αS deposits, and motor dysfunction.7., 8. Therefore, αS, and in particular αS aggregation, appears to be intimately associated with the pathogenesis of PD. Recently, αS was shown to be covalently modified by dopamine under oxidizing conditions, leading to an increased oligomerization rate of the protein.9 This observation provides an appealing explanation for why dopaminergic neurons are preferentially damaged in PD, and for the plethora of data suggesting a link between oxidative stress and PD.

The normal function of αS remains poorly understood. The earliest identification of the protein reported an eponymous synaptic and perinuclear localization.10 Synaptic localization has been confirmed repeatedly,11 while perinuclear localization remains controversial. In the synapse, αS appears to localize to the vicinity of synaptic vesicles, and conflicting reports regarding direct vesicle association suggest that the protein is probably involved in reversible interactions with synaptic vesicles. The presence of seven imperfect 11-mer repeats in the αS sequence that bear a strong resemblance to 11-mer repeats observed in the exchangeable apolipoproteins12., 13. supports this hypothesis, as reversible lipid interactions are a hallmark of apolipoprotein function. The amphipathic nature and reversible lipid interactions of αS strongly suggest that the protein binds only to lipid surfaces, and does not insert into the interior of membranes. αS is instrinsically unstructured when free in solution,13., 14. but undergoes a structural transition to a highly helical state in the presence of either brain-derived or synthetic lipid vesicles.15., 16.

In addition to interacting with lipids, αS is presumed, like other intrinsically unstructured proteins, to interact with one or more protein partners. We have shown that when αS is in its lipid-bound conformation the C-terminal tail of the protein does not associate with the lipid membrane, and is therefore a likely site for protein–protein interactions.14 A number of reports describe interactions between αS and other proteins, including PLD2,17 tau,18 14–3–3 proteins, protein kinase Cα, BAD,19 synphilin20 and tubulin.21 αS is also reportedly phosphorylated by Src family kinases,22 casein kinases23 and G protein coupled receptor kinases.24 Based on these and other observations, a role has been suggested for αS in synaptic vesicle and/or fatty acid25 transport or regulation, phospholipid metabolism, and chaperone functions. Mouse knockouts of αS result in subtle defects in synaptic vesicle pool size and recycling26., 27. confirming a synaptic vesicle-associated function, but shedding little light on the exact nature of this function. αS is a member of a larger protein family, including β and γ-synuclein. β-Synuclein, which is also highly expressed in the brain, may be able to compensate for a lack of αS, and may also affect aS aggregation.28 Studies of double or triple knockouts have not been reported. αS also appears to play a role in song learning in birds.12

Based on the amphipathic nature of the αS sequence a model has been proposed for the helical structure of lipid-bound αS.15 In this model (henceforth referred to as the five helix model) a helical pinwheel representation of the protein was used to delineate five amphipathic α-helical segments, four of which fit the apolipoprotein class A2 helix motif.29 Five separate helices were required because highly amphipathic α-helices could only be modeled over relatively short stretches of the αS sequence. Although circular dichroism15., 16. and our previous NMR data14 clearly support the presence of helical structure in lipid-bound αS, the details of the five helix model have remained untested.

Here, we use high-resolution solution state NMR to probe the structural properties of monomeric αS bound to lipid-mimetic detergent micelles at a level of detail sufficient to evaluate the five helix model. Our results are inconsistent with the presence of five separate helical segments. Instead, we observe evidence for two longer helical stretches, interrupted by a short break. Application of a quantitative algorithm for finding lipid binding helices in proteins30., 31. also results in the identification of two helical regions. Modeling of αS as a single uninterrupted α-helix reveals a rotating hydrophobic surface that is not suitable for binding a lipid bilayer. Thus, the break in the helical αS structure may be required to maintain the alignment of the hydrophobic surfaces of the two shorter helices. However, the two helices generated by the break that we observe remain imperfectly amphipathic, and may adopt an alternative α11/3 helical conformation,32 completing three full turns over the span of 11 residues (contrasted with five turns over 18 residues for an ideal α-helix) and allowing the sequence to form well-demarcated hydrophobic and hydrophilic faces. Calculated lipid binding affinities30., 31. of the α11/3 and ideal α-helical conformations for the two helices reveals that the first helix has equivalent affinity in either conformation, while the second helix has a significantly greater affinity in the α11/3 conformation.

We discuss several possibilities for the cause and effect of the break that we observe in the helical structure of αS. The effect of the break may be to provide a hinge that allows the protein to bind to a curved surface, or to make functionally important residues at or near the break accessible. The cause of the break may be the need to realign the twisting hydrophobic face of the αS helix, which is in turn caused by the 11-mer repeats of the protein, or the break may be caused by the appearance of a nearby tyrosine in the protein sequence. Alternately, the break may in fact be an artifact of the very high curvature of the micelle surface, and may not be required for binding lipid surfaces of lower curvatures. In this case, a long unbroken αS helix would have to adopt the α11/3 conformation32 to bind a lipid surface.

We use a search of protein sequence databases to document the presence of αS-like 11-mer repeats in other reversibly lipid binding proteins. This observation allows for a partial annotation of a number of proteins of unknown function, and supports the idea that a structural feature of these 11-mer repeats may play an important functional role in the lipid interactions of these proteins. Possible mechanisms that might mediate such a role include: limiting the length of lipid bound α-helices through the twisting nature of the hydrophobic faces formed by 11-mer repeats in order to generate flexible hinge regions for binding curved lipid surfaces; forming α11/3 helices, which may be associated with an energetic cost33 that could reduce the stability of the resulting protein lipid complexes and thereby facilitate reversible lipid binding; forming right-handed coiled coil structures34., 35., 36., 37., 38. in the lipid-free state, thus providing a hydrophobic surface that is suitable for lipid interactions, but can be sequestered in the absence of lipids.

Section snippets

Results

In previous work we showed that the conformational change induced in αS upon lipid binding is recapitulated in the presence of SDS micelles, and that in both micelle and lipid vesicle bound αS the C-terminal residues of the protein (beyond position 103) do not interact with the micelle or lipid vesicle.14 We have used limited proteolysis as a further probe, to evaluate which regions of αS are structured and which are not in the presence of micelles and lipid vesicles. Helical structure and/or

Discussion

Despite the intense interest in αS generated by its role in Parkinson's disease, little detailed information is available on the structure of this protein. When free in solution in vitro, αS is largely unstructured.13., 14. Although one region of the protein exhibits residual helical structure that may play a role in modulating the aggregation kinetics of αS fibril formation,46 it is unlikely that the structural properties of the free form of the protein are directly related to its normal

Conclusions

Our results suggest that 11-mer repeats are likely to play an important role in the lipid binding function of αS and a number of other proteins in one of several ways. One possibility suggested by our analysis is that 11-mer repeats may lead to a non-canonical conformation, the α11/3 helix,32 which differs slightly in pitch from the ideal α-helix. An α11/3 helix would likely be associated with a degree of conformational strain because of the deviation from the energetically favorable ideal

Methods

Protein samples were prepared as described.14 Lipid vesicles were prepared by mixing appropriate amounts of freshly procured 1-palmitoyl 2-oleoyl phosphatidyl choline and phosphatidic acid in chloroform (Avanti Polar Lipids). The mixture was dried on a Rotovap, subjected to overnight lyophilization to remove traces of organic solvent, and resuspended in sample buffer. To prepare SUVs, the resuspended lipid mixture was sonicated on ice and ultracentrifuged at 60,000 rpm (Sorval S120-AT2 rotor)

Supplementary Files

Acknowledgements

This work was supported, in part, by the NIA, National Institutes of Health, through grant AG19391 (to D.E.) and by a gift from Herbert and Ann Siegel (to D.E.). We thank Dr Peter Lansbury (Harvard Medical School) for valuable discussions and for generously providing us with the α-synuclein gene construct, Dr Min Lu (Weill Medical College of Cornell University) for valuable discussions, reading the manuscript, and assistance with the analytical ultracentrifugation measurements, Dr Art Palmer

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