Structure and dynamics of the extended-helix state of alpha-synuclein: Intrinsic lability of the linker region.

The Parkinson's protein alpha-synuclein binds to synaptic vesicles in vivo and adopts a highly extended helical conformation when binding to lipid vesicles in vitro. High-resolution structural analysis of alpha-synuclein bound to small lipid or detergent micelles revealed two helices connected by a non-helical linker, but corresponding studies of the vesicle-bound extended-helix state are hampered by the size and heterogeneity of the protein-vesicle complex. Here we employ fluorinated alcohols (FAs) to induce a highly helical aggregation-resistant state of alpha-synuclein in solution that resembles the vesicle-bound extended-helix state but is amenable to characterization using high-resolution solution-state NMR. Analysis of chemical shift, NOE, coupling constant, PRE and relaxation measurements shows that the lipid-binding domain of alpha-synuclein in FA solutions indeed adopts a single continuous helix and that the ends of this helix do not come into detectable proximity to each other. The helix is well ordered in the center, but features an increase in fast internal motions suggestive of helix fraying approaching the termini. The central region of the helix exhibits slower time scale motions that likely result from flexing of the highly anisotropic structure. Importantly, weak or missing short- and intermediate-range NOEs in the region corresponding to the non-helical linker of micelle-bound alpha-synuclein indicate that the helical structure in this region of the protein is intrinsically unstable. This suggests that conversion of alpha-synuclein from the extended-helix to the broken-helix state represents a functionally relevant structural transition.

Residual structure, backbone dynamics, and interactions within the synuclein family.

The human synuclein protein family includes alpha-synuclein, which has been linked to both familial and sporadic Parkinson's disease, and the highly homologous beta and gamma-synuclein. Mutations in alpha-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 beta and gamma-synuclein, and any neurodegenerative disease has been established, and it is generally considered that these proteins are not highly pathogenic. In addition, beta and gamma-synuclein are reported to aggregate less readily than alpha-synuclein in vitro. Indeed, beta-synuclein has been reported to protect against alpha-synuclein aggregation in vitro, as well as alpha-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 gamma-synuclein closely resembles alpha-synuclein in its free-state residual secondary structure, consistent with the more similar propensities of the two proteins to aggregate in vitro. beta-Synuclein, however, differs significantly from alpha-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 beta and gamma-synuclein show less extensive transient long-range structure than that observed in alpha-synuclein. These results raise questions regarding the role of secondary structure propensities and transient long-range contacts in directing synuclein aggregation reactions.

Secondary structure and dynamics of micelle bound beta- and gamma-synuclein.

We have used solution state NMR spectroscopy to characterize the secondary structure and backbone dynamics of the proteins beta- and gamma-synuclein in their detergent micelle-bound conformations. Comparison of the results with those previously obtained for the Parkinson's disease-linked protein alpha-synuclein shows that structural differences between the three homologous synuclein family members are directly related to variations in their primary amino acid sequences. An 11-residue deletion in the lipid-binding domain of beta-synuclein leads to the destabilization of an entire segment of the micelle-bound helical structure containing the deletion site. The acidic C-terminal tail region of gamma-synuclein, which displays extensive sequence divergence, is more highly disordered than the corresponding regions in the other two family members. The observed structural differences are likely to mediate functional variations between the three proteins, with differences between alpha- and beta-synuclein expected to revolve around their lipid interactions, while differences in gamma-synuclein function are expected to result from different protein-protein interactions mediated by its unique C-terminal tail.

NMR mapping of copper binding sites in alpha-synuclein.

Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein.