Hai-Young Kim

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinsons disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.

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 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.

Phosphorylation Drives a Dynamic Switch in Serine/Arginine-Rich Proteins

Serine/arginine-rich (SR) proteins are important players in RNA metabolism and are extensively phosphorylated at serine residues in RS repeats. Here, we show that phosphorylation switches the RS domain of the serine/arginine-rich splicing factor 1 from a fully disordered state to a partially rigidified arch-like structure. Nuclear magnetic resonance spectroscopy in combination with molecular dynamics simulations revealed that the conformational switch is restricted to RS repeats, critically depends on the phosphate charge state and strongly decreases the conformational entropy of RS domains. The dynamic switch also occurs in the 100 kDa SR-related protein hPrp28, for which phosphorylation at the RS repeat is required for spliceosome assembly. Thus, a phosphorylation-induced dynamic switch is common to the class of serine/arginine-rich proteins and provides a molecular basis for the functional redundancy of serine/arginine-rich proteins and the profound influence of RS domain phosphorylation on protein-protein and protein-RNA interactions.

Conserved core of amyloid fibrils of wild type and A30P mutant α-synuclein

The major component of neural inclusions that are the pathological hallmark of Parkinsons disease are amyloid fibrils of the protein α‐synuclein (aS). Here we investigated if the disease‐related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two‐dimensional proton‐detected high‐resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of β‐strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano‐second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation‐induced change in the conformation of aS aggregates.

Dissociation of Amyloid Fibrils of α‐Synuclein in Supercooled Water

Several neurodegenerative diseases, including Alzheimer s, Creutzfeldt–Jakob, and Parkinson s disease, are associated with the formation of amyloid fibrils. Amyloid fibrils have a b-sheet-rich molecular architecture called a cross-b structure. The b-sheet conformation imparts extremely high thermodynamic stability and remarkable physical properties to amyloid fibrils. They are highly resistant to hydrostatic pressure and high temperature, whereas protofibrils and earlier aggregates are more sensitive to these extreme conditions. The stability of mature amyloid fibrils exceeds that of globular proteins, thus suggesting that they may represent the global minimum in terms of free energy. In addition, they have a strength comparable to that of steel. Nature exploits these unusual properties of amyloidgenic structures for a variety of physiological functions. Moreover, fibrillar peptide structures might have great potential as structural or structuring elements in nanotechnology applications. The native state of proteins can be unfolded both by high temperature and by cooling. Cold denaturation is predicted by the Gibbs–Helmholtz Equation, and attributed to specific interactions between nonpolar protein groups and water: tightly packed structures unfold at sufficiently low temperature to expose internal nonpolar groups to the water. Direct observation of cold denaturation is generally hard to achieve in the absence of denaturant, extreme pH values, or mutations, as the transition temperature for most proteins is well below 0 8C. Freezing, however, can be avoided down to temperatures as low as 20 8C by careful supercooling of small sample volumes. Nevertheless, this is generally not sufficiently cold to induce denaturation in stable, native proteins. Here we demonstrate that amyloid fibrils of the protein asynuclein (aS), which constitute the insoluble aggregates found in brains of patients suffering from Parkinson s disease, are highly sensitive to low temperature. Despite their remarkable stability to hydrostatic pressure and high temperatures, mature amyloid fibrils of aS rapidly dissociate in supercooled water at 15 8C. N-Labeled aS amyloid fibrils were prepared in vitro by incubating 0.1 mm freshly prepared N-labeled aS in 20 mm tris(hydroxymethyl)aminomethane (Tris) and 100 mm NaCl at pH 7.4. Incubation was carried out under continuous stirring at 37 8C for up to 14 days until a steady state was reached, as judged by thioflavin-T (ThT) fluorescence. Matured fibrils were pelleted by centrifugation at 215000g for 2 h and then resuspended in 50 mm phosphate buffer. Transmission electron micrographs showed regular fibrils with a diameter of approximately 40 nm (Figure 1a). A strong ThT fluorescence signal was detected for the fibrils (see the Supporting Information). Previous X-ray diffraction and solid-state NMR measurements have shown that amyloid fibrils of aS adopt a cross-b structure. No cross-peaks were visible in the H-N HSQC spectra, which is in agreement with the large molecular weight of amyloid fibrils and their associated fast relaxation (Figure 1a).

Cold denaturation of a protein dimer monitored at atomic resolution

Protein folding and unfolding are crucial for a range of biological phenomena and human diseases. Defining the structural properties of the involved transient species is therefore of prime interest. Using a combination of cold denaturation with NMR spectroscopy, we reveal detailed insight into the unfolding of the homodimeric repressor protein CylR2. Seven three-dimensional structures of CylR2 at temperatures from 25 °C to -16 °C reveal a progressive dissociation of the dimeric protein into a native-like monomeric intermediate followed by transition into a highly dynamic, partially folded state. The core of the partially folded state seems critical for biological function and misfolding.

Cold-induced changes in the protein ubiquitin.

Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the β-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation.