David Eliezer

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

We have used NMR spectroscopy and limited proteolysis to characterize the structural properties of the Parkinsons disease-related protein alpha-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 alphaS as an ideal alpha-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 alphaS structure would need to adopt an unusual, slightly unwound, alpha11/3 helix conformation (three complete turns per 11 residues). The break we observe in the alphaS helix may allow the protein to avoid this unusual conformation by adopting two shorter stretches of typical alpha-helical structure. However, a quantitative analysis suggests the possibility that the alpha11/3 conformation may in fact exist in lipid-bound alphaS. We discuss how structural features of helical 11-mer repeats could play a role in the reversible lipid binding function of alpha-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.

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Background: The oligomeric state of α-syn in vivo remains unknown. Results: α-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native α-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric α-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease. Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107–110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797–17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

Biophysical characterization of intrinsically disordered proteins

The challenges associated with the structural characterization of disordered proteins have resulted in the application of a host of biophysical methods to such systems. NMR spectroscopy is perhaps the most readily suited technique for providing high-resolution structural information on disordered protein states in solution. Optical methods, solid state NMR, ESR and X-ray scattering can also provide valuable information regarding the ensemble of conformations sampled by disordered states. Finally, computational studies have begun to assume an increasingly important role in interpreting and extending the impact of experimental data obtained for such systems. This article discusses recent advances in the applications of these methods to intrinsically disordered proteins.

Structural Basis for Recognition of Diubiquitins by NEMO

NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB 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 geometry for mutual recognition of NEMO and diubiquitins.

Biophysics of Parkinson's disease: structure and aggregation of alpha-synuclein.

Parkinsons disease (PD) is a slowly progressive movement disorder that results from the loss of dopaminergic neurons in the substantia nigra, a small area of cells in the mid-brain. PD is a multifactorial disorder with unknown etiology, in which both genetic and environmental factors play important roles. Substantial evidence links alpha-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic PD. Rare familial cases of PD are associated with missense point mutations in alpha-synuclein, or with the hyper-expression of the wild type protein due to its gene duplication/triplication. Furthermore, alpha-synuclein was identified as the major component of amyloid fibrils found in Lewy body and Lewy neurites, the characteristic proteinaceous deposits that are the diagnostic hallmarks of PD. alpha-Synuclein is abundant in various regions of the brain and has two closely related homologs, beta-synuclein and gamma-synuclein. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces alpha-synuclein adopts a highly helical structure that is believed to mediate its normal function(s). A number of different conformational states of alpha-synuclein have been observed. Besides the membrane-bound form, other critical conformations include a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. A number of intrinsic and extrinsic factors that either accelerate or inhibit the rate of alpha-synuclein aggregation and fibrillation in vitro are known. There is a strong correlation between the conformation of alpha-synuclein (induced by various factors) and its rate of fibrillation. The aggregation process appears to be branched, with one pathway leading to fibrils and another to oligomeric intermediates that may ultimately form amorphous deposits. The molecular basis of Parkinsons disease appears to be tightly coupled to the aggregation of alpha-synuclein and the factors that affect its conformation. This review focuses on the contributions of Prof. Anthony L. Fink to the field and presents some recent developments in this exciting area.

Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of α-synuclein

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.

Phosphorylation at S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization and influences synuclein-membrane interactions.

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 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 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 fibrillization in 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.

Identification of a copper-binding metallothionein in pathogenic mycobacteria.

A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially binds up to six Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the genes expression in Mtb up to 1,000-fold above normal expression. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionein (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.

Helix periodicity, topology, and dynamics of membrane‐associated α‐Synuclein

The protein α‐Synuclein (aS) is a synaptic vesicle‐associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinsons disease. The normal function of aS depends on a membrane‐associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane‐binding domain of aS is helical and suggested that it may exhibit an unusual structural periodicity. Here we present a study of the periodicity, topology, and dynamics of detergent micelle‐bound aS using paramagnetic spin labels embedded in the micelle or attached to the protein. We show that the helical region of aS completes three full turns every 11 residues, demonstrating the proposed 11/3 periodicity. We also find that the membrane‐binding domain is partially buried in the micelle surface and bends toward the hydrophobic interior, but does not traverse the micelle. Deeper submersion of certain regions within the micelle, including the unique lysine‐free sixth 11‐residue repeat, is observed and may be functionally important. There are no long‐range tertiary contacts within this domain, indicating a highly extended configuration. The backbone dynamics of the micelle‐bound region are relatively uniform with a slight decrease in flexibility observed toward the C‐terminal end. These results clarify the topological features of aS bound to membrane‐mimicking detergent micelles, with implications for aS function and pathology.

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

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.