Claudio O. Fernández

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.

NMR of α-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

The aggregation of α‐synuclein is characteristic of Parkinsons 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.

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.

Structural Properties of Pore-Forming Oligomers of α-Synuclein

Soluble oligomers are potent toxins in many neurodegenerative diseases, but little is known about the structure of soluble oligomers and their structure-toxicity relationship. Here we prepared on-pathway oligomers of the 140-residue protein alpha-synuclein, a key player in Parkinsons disease, at concentrations an order of magnitude higher than previously possible. The oligomers form ion channels with well-defined conductance states in a variety of membranes, and their beta-structure differs from that of amyloid fibrils of alpha-synuclein.

Site-specific interactions of Cu(II) with α and β-synuclein: bridging the molecular gap between metal binding and aggregation.

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.

Bioinorganic Chemistry of Parkinson's Disease: Structural Determinants for the Copper-Mediated Amyloid Formation of Alpha-Synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

Structural and mechanistic basis behind the inhibitory interaction of PcTS on alpha-synuclein amyloid fibril formation.

The identification of aggregation inhibitors and the investigation of their mechanism of action are fundamental in the quest to mitigate the pathological consequences of amyloid formation. Here, characterization of the structural and mechanistic basis for the antiamyloidogenic effect of phthalocyanine tetrasulfonate (PcTS) on α-synuclein (AS) allowed us to demonstrate that specific aromatic interactions are central for ligand-mediated inhibition of amyloid formation. We provide evidence indicating that the mechanism behind the antiamyloidogenic effect of PcTS is correlated with the trapping of prefibrillar AS species during the early stages of the assembly process. By using NMR spectroscopy, we have located the primary binding region for PcTS to a specific site in the N terminus of AS, involving the amino acid Tyr-39 as the anchoring residue. Moreover, the residue-specific structural characterization of the AS-PcTS complex provided the basis for the rational design of nonamyloidogenic species of AS, highlighting the role of aromatic interactions in driving AS amyloid assembly. A comparative analysis with other proteins involved in neurodegenerative disorders reveals that aromatic recognition interfaces might constitute a key structural element to target their aggregation pathways. These findings emphasize the use of aggregation inhibitors as molecular probes to assess structural and toxic mechanisms related to amyloid formation and the potential of small molecules as therapeutics for amyloid-related pathologies.

Structural characterization of α‐synuclein in an aggregation prone state

The relation of α‐synuclein (αS) aggregation to Parkinsons disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of αS in an aggregation‐prone state, we studied the structural properties of αS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that αS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of αS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu‐rich C‐terminal region, an increased probability for proximity between the NAC‐region and the C‐terminal region and a lower probability for interactions between the N‐ and C‐terminal regions.

Correlation of Amyloid Fibril β-Structure with the Unfolded State of α-Synuclein

Many human neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, are associated with the deposition of proteinaceous aggregates known as amyloid fibrils. 2] Surprisingly, proteins with very different amino acid sequences and three-dimensional structures aggregate into amyloid fibrils that share common characteristics, such as a similar morphology and a specific b-sheet-based molecular architecture. This suggests that the ability to fibrillate is an intrinsic property of a polypeptide chain and that the native structure is not necessarily the only ordered structure that each protein can assume. An additional common property of aggregation into amyloid fibrils is the presence of partially or fully unfolded states of the misfolding proteins. For proteins that are natively folded, unfolded states are present during biosynthesis, or as a result of proteolytic degradation. Whereas there is increasing knowledge about the factors that drive aggregation, the structural characteristics of aggregation-prone states and the molecular details that determine the arrangement of misfolded proteins in amyloid fibrils are still only understood in outline. Is a high population of a specific conformation in a denatured state required for the efficient generation of amyloid fibrils? What is the relation between the properties of the unfolded state and the b-structure in amyloid fibrils? Are differences in the morphologies of the resulting fibrils associated with differences in the structures of the precursor states? Here we demonstrate by a combination of solution-state and solid-state NMR spectroscopy that the structure of amyloid fibrils is directly correlated to the conformational properties of the unfolded state. Similar or even identical spectroscopic probes are used in solution-state and solidstate NMR, allowing a detailed comparison of the structure of proteins in their unfolded and fibrillar states. a-Synuclein (aS) is the major component of abnormal aggregates in the brains of patients with Parkinson’s disease, the most common neurodegenerative movement disorder. When bound to membranes, the 140-amino acid protein adopts an a-helical conformation. In solution, monomeric aS was found to be highly dynamic and was classified as natively unfolded. We and others recently showed that, despite its high flexibility, native aS adopts an ensemble of conformations stabilized by long-range interactions. Conditions that destabilize long-range interactions lead to conformations that associate readily, resulting in aggregation in vitro. The structure of aS in amyloid fibrils was studied by various techniques, and these studies showed that the cores of aS fibrils are formed by residues ~34 to ~101, comprising at least four bstrands, whereas the Nand C-terminal domains remain disordered. When the temperature of the solution is reduced sufficiently without freezing, proteins can be cold denatured without the need for chemicals that would potentially interfere with the ensemble of conformations present in solution. In addition, ACHTUNGTRENNUNGinterconversion between different conformations that causes averaging of spectroscopic probes at higher temperatures is slowed down (see Figure S1 in the Supporting Information). Here we have used supercooling to remove transient longrange interactions in aS. Using thin glass capillaries, we reduced the temperature in the protein solution to 15 8C without freezing. At 15 8C the hydrodynamic radius of aS was ACHTUNGTRENNUNGsignificantly increased and adopted a value similar to that observed in the presence of 8m urea at 2 8C, 15 8C and 47 8C (Figure 1A). At high temperature, on the other hand, the hydrodynamic radius reached a maximum value that was further increased upon addition of urea, suggesting that long-range ACHTUNGTRENNUNGinteractions are more efficiently removed at 15 8C than at 57 8C. We then determined the sequence-specific assignment of backbone resonances of aS by 3D triple-resonance NMR experiments and observed paramagnetic relaxation enhancement of amide protons due to the presence of a nitroxide spin label attached to residue 18. The reduction in line broadening at 15 8C at the C terminus indicates the removal of transient long-range interactions (Figure S3), in agreement with the increase in hydrodynamic radius. NMR chemical shifts, especially of C and C’ atoms, are very sensitive probes of secondary structure in proteins. These shifts showed small but distinct deviations from random coil values for aS in solution at 15 8C (Figure 1B). For residues 1– 38, the signs of secondary NMR chemical shifts alternated; this indicated the absence of a propensity for either helical or bstructure. Residues 38–98, which form the cores of amyloid fibrils, as well as the acidic C-terminal domain, showed predominantly negative secondary chemical shifts. Whereas negative secondary chemical shifts are characteristic of either b-structure or polyproline helix-like conformations, large JACHTUNGTRENNUNG(H,H) scalar couplings are only found in extended b-structures. In the C-terminal domain of aS, the JACHTUNGTRENNUNG(H,H) values were mostly below average, in agreement with the presence of several proline residues. Only for residues 38–98 were negative secondary chemical shifts and above-average J ACHTUNGTRENNUNG(H,H) couplings observed; this suggests that the central domain of aS transiently populates the b-region in the Ramachandran plot. [a] H.-Y. Kim, Dr. H. Heise, Dr. M. Baldus, Dr. M. Zweckstetter Department of NMR-based Structural Biology Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gçttingen (Germany) Fax: (+49)551-201-2202 E-mail : mzwecks@gwdg.de [b] Dr. C. O. Fernandez Instituto de Biolog;a Molecular y Celular de Rosario Universidad Nacional de Rosario Suipacha 531, S2002 LRK Rosario (Argentina) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Exploring the structural details of Cu(I) binding to α-synuclein by NMR spectroscopy.

The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS.