In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein α-synuclein (αS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, αS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric αS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro , release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of αS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

The aggregation of α-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS–Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of α-synuclein (α-syn) in Parkinson disease. Herein we demonstrate that α-syn is phosphorylated at S87in vivoand within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated α-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P α-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric α-syn. These studies demonstrated that phosphorylation at S87 expands the structure of α-syn, increases its conformational flexibility, and blocks its fibrillizationin vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of α-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in α-syn normal biology.

The aggregation of α‐synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140‐aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of α‐synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with α‐synuclein by NMR and assigned the binding site to C‐terminal residues 109–140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 → +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by ∼104 and the rate of monomer addition ∼40‐fold. Significant secondary structure is not induced in monomeric α‐synuclein by polyamines at 15°C. Instead, NMR reveals changes in a region (aa 22–93) far removed from the polyamine binding site and presumed to adopt the β‐sheet conformation characteristic of fibrillar α‐synuclein. We conclude that the C‐terminal domain acts as a regulator of α‐synuclein aggregation.