Long‐Range Distances in Amyloid Fibrils of α‐Synuclein from PELDOR Spectroscopy

Abstract

a-Synuclein (aS), a small protein containing 140 amino acids, undergoes self-assembly into amyloid fibrils and plaques (Lewy bodies), which are pathological hallmarks of Parkin-sons disease (PD) as well as other neurodegenerative diseases. [1] While oligomeric species of aS are considered to exert the neurotoxic activity [2] that can be rescued by reducing those oligomers either by diversion to smaller oligomers [3] or acceleration of fibril formation, cell-to-cell transmission in nontransgenic mice points to a direct role of the fibrils in spreading the disease from peripheral to central neurons. [4] Understanding the molecular interactions that lead to mis-folding strongly relies on the availability of suited biophysical methods that can access the structure of these states. For the monomeric form, magnetic resonance techniques gave evidence for a natively disordered yet partially folded protein [5a,b] that upon binding to lipid vesicles adopts an a-helical structure. [6-8] When aS is aggregated into fibrils, X-ray and electron diffraction studies [9a,b] revealed that it arranges in a classical cross-b conformation, where the individual b-sheets arrange perpendicularly to the fibril axis with spacing of 4.7-4.8 Š along the fibril axis and 10-11 Š perpendicular to the axis. In particular, aS stacks in a parallel, in-register arrangement as revealed by continuous-wave (CW) electron paramagnetic resonance (EPR) [10] and more recently supported by solid-state nuclear magnetic resonance (ss-NMR) spectroscopy. [11] The structural features of monomeric aS consist of: 1) an amphipathic N-terminal region (residues 1-60), 2) a hydro-phobic central domain, known as a non-Ab component (NAC) region (residues 61-95), and 3) a highly negatively charged C-terminal region (residues 96-140). [12] Studies from ss-NMR and CW-EPR spectroscopy have shown that the b-sheet core region is located within the central NAC domain [10, 13a-d] and extended toward the N-terminus [14a,b] whereas the C-terminal region appears less ordered. Figure 1 represents the amino acid sequence of human aS as well as b-strands within the NAC region assigned by different ss-NMR studies. All these studies identified at least five b-strands that could fold to generate the core unit of the fibril structure. One model of the three-dimensional fold of the fibril consisting of five b-sheets per monomer has been proposed [13b] based on ss-NMR and cryo-electron microscopic data. However, long-range restraints at the molecular level in support of this model are still missing. Recently, we have Figure 1. Top: Amino acid sequence of human aS in the NAC region. Mutation positions are marked in red. Bottom: Arrows indicate b-strand regions as identified by ss-NMR spectroscopy. a) Light blue, 13