␣-Synuclein (␣-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related ␣-synulceinopathies. However, whether phosphorylation promotes or inhibits ␣-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of ␣-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 ␣-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated ␣-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of ␣-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 ␣-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 ␣-syn aggregation and toxicity in PD and related ␣-synulceinopathies.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of ␣-synuclein (␣-syn) in Parkinson disease. Herein we demonstrate that ␣-syn is phosphorylated at S87 in vivo and 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 ␣-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 ␣-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 membranebinding properties of monomeric ␣-syn. These studies demonstrated that phosphorylation at S87 expands the structure of ␣-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of ␣-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 ␣-syn normal biology.

SUMMARY NEMO is the regulatory subunit of the IκB kinase (IKK) in NF-κB activation and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch while the distal ubiquitin mostly employs the C-terminal arm including the K63-linkage residue. These studies uncover the energetics and the common U-shaped geometry for mutual recognition of NEMO and diubiquitins.

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein R-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K R-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K R-synuclein formed insoluble fibrils in Vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K R-synuclein fibrils possess a typical amyloid ultrastructure. E46K R-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of R-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type R-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in Vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of R-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT R-synuclein that are observed in Vitro.Parkinson's disease (PD 1 ) is a progressive neurodegenerative disorder characterized by resting tremor, bradykinesia, rigidity, and postural instability due to the selective loss of dopaminergic neurons within the substantia nigra (2, 3). While nearly all cases of PD are idiopathic, rare forms of autosomal dominant PD have been linked to the point mutations A53T (4), A30P (5), and E46K (1) in R-synuclein, a presynaptic protein believed to be involved in synaptic vesicle trafficking (6,7). Linkage between idiopathic PD and R-synuclein is suggested by the discovery that Lewy bodies (LB), intraneuronal cytoplasmic inclusions in the substantia nigra that are the pathological hallmark of PD, are composed primarily of fibrillar R-synuclein (8).Monomeric R-synuclein is natively unfolded in solution (9), but assumes -sheet character as it aggregates through a series of intermediate, metastable oligomeric states (termed protofibrils) to a stable fibrillar conformation (10). In Vitro studies have shown that both the A53T and A30P mutations in R-synuclein alter the kinetics of fibrillization; the rate is increased for the A53T variant and retarded by the A30P substitution (11,12). However, in both cases the rates of formation of pre...

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, β-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v∕v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type αS and the C-terminal truncation mutant αS1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type αS, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that αS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced αS aggregation both in vitro and in vivo.is one of a number of synucleopathies in which aggregation of the protein α-Synuclein (αS) is linked to pathogenesis (1). αS is intrinsically disordered, but in the presence of lipid or detergent vesicles or micelles, adopts a highly helical structure in which its N-terminal region is membrane-bound and the C-terminal tail remains predominantly free and unstructured (2, 3). Although most PD cases are sporadic or idiopathic, three point mutations of α-Synuclein-A53T, A30P, and E46K-are associated with familial and early-onset disease (see Review (4)).In addition to its free and membrane-bound states, αS adopts partially structured intermediate conformations under low-pH or high-temperature conditions (5). A folding intermediate has also been detected at low [TFE] (6). Conditions favoring the formation of these intermediates also promote amyloid fibril growth, possibly implicating intermediate conformers as key species in the aggregation pathways.Here, we examine TFE-induced monomer conformational changes, oligomerization, and fibrillization in detail for wild-type (WT) αS, C terminally truncated WT αS (αS102), and the PDassociated αS mutants A30P, A53T, and E46K, expanding upon previous studies by Munishkina, et. al. (6) and Li, et. al. (7). This research also complements our previous fluorescence correlatio...

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Parkinson's disease (PD) is associated with the deposition of fibrillar aggregates of the protein α-synuclein (αS) in neurons. Intramolecular contacts between the acidic C-terminal tail of αS and its N-terminal region have been proposed to regulate αS aggregation, and two originally described PD mutations, A30P and A53T, reportedly reduce such contacts. We find that the most recently discovered PD-linked αS mutation E46K, which also accelerates the aggregation of the protein, does not interfere with C-terminal-to-N-terminal contacts and instead enhances such contacts. Furthermore, we do not observe a substantial reduction in such contacts in the two previously characterized mutants. Our results suggest that Cterminal-to-N-terminal contacts in αS are not strongly protective against aggregation, and that the dominant mechanism by which PD-linked mutations facilitate αS aggregation may be altering the physicochemical properties of the protein such as net charge (E46K) and secondary structure propensity (A30P and A53T).

Alpha-synuclein (aS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of aS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased aS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side-chains eliminates electrostatic repulsion in the C-terminal tail of aS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C-terminal tail and the NAC (non-amyloid-b component) region are maintained and may lead to the formation of a globular domain. Transient long-range contacts between the C-terminus of the protein and regions N-terminal to the NAC region are also preserved. Thus, the release of long-range contacts does not play a role in the increased aggregation of aS at low pH, which we instead attribute to the increased hydrophobicity of the protein.