The Clustering and Spatial Arrangement of β-Sheet Sequence, but Not Order, Govern α-Synuclein Fibrillogenesis

Abstract

The intrinsically unstructured protein α-synuclein (aS) is prone to misfold into cytotoxic β-sheet-rich oligomers and amyloid fibrils that underlie the pathogenesis of Lewy body diseases such as Parkinson's disease. An important, recognized fibrillogenesis parameter is amino acid content, whereas the influence of amino acid sequence distribution is not as well understood. The fibril core of aS encompasses five regions of high β-sheet propensity, termed β1-β5. Using four aS variants with identical amino acid composition but rearranged pseudorepeat motifs, we show that β2-β5 sequence clustering, but not order, is important for efficient fibrillogenesis. For molecular species progressing towards the fibrillar state, order invariably increases; i.e., the spatial arrangement of sequence elements becomes restricted. By introducing disulfide bonds in a fibril structure-based manner, it is demonstrated that a successful protofibril-to-fibril conversion is dependent upon the spatial arrangement of sequence elements of high β-sheet propensity. Moreover, a disulfide-linked aS dimer is shown to fibrillize rapidly. We propose that a conformational search underlies the emergence of a fibrillar aS nucleus that is directed by gaps in sequence between β-sheet regions and the accessible range of spatial β-sheet arrangements in soluble, prefibrillar oligomers. Based on the universal cross-β sheet structure of amyloid fibrils, these principles are expected to apply to a wide range of amyloidogenic proteins.

Driving Forces and Structural Determinants of Steric Zipper Peptide Oligomer Formation Elucidated by Atomistic Simulations

“…28 Finally, the oligomerization of larger peptides and full-length proteins might obey similar principles but usually needs to be accompanied by a substantial change or loss of intra-peptide interactions. Moreover, aggregation is facilitated by transient misfolding or unfolding events of particular aggregation-prone segments, which populate amyloid competent conformers, such as hairpin motifs or intra-molecular β-strand stacks in natively disordered peptides 41,45,54,90 or accessible self-complementary stretches on protein surfaces. 22,23,67 Recent findings furthermore suggest that while oligomer formation might be facilitated by small segments and their local properties, the eventual maturation toward fibrillar structure will involve a remodeling stage to further incorporate and accommodate residues into the β-strands.…”

α-Synuclein Populates Both Elongated and Broken Helix States on Small Unilamellar Vesicles

“…Following ion exchange chromatography using HiTrap Q-Sepharose (GE Healthcare), any residual DNA was digested with 500 Kunitz units of DNase I (bovine pancreas; EMD Chemicals, Inc.) for 1 h at 37°C to exclude spectral contamination in concentration measurements. The desired intramolecularly disulfide-bonded proteins, ␣S(Cys 11 -Cys 81 ) and ␣S(Cys 11 -Cys 83 ), were then separated from covalently linked protein oligomers by gel filtration employing a Sephacryl S-100 HR 26/60 column (GE Healthcare) as described previously in detail (34). Sample purity was verified by SDS-PAGE (supplemental Fig.…”

“…interconversions of vesicle-bound ␣S segments may bear relevance to accessing its misfolding pathway (32,33), which initially exhibits characteristics of a conformational search (34). At a given time, protein structures are generally characterized by a single well defined conformation, and the existence of multiple parallel ␣S conformations would be an interesting deviation from this paradigm.…”

Computational Study on Oligomer Formation of Fibril-forming Peptide of α-Synuclein

“…[3][4][5] Numerous biophysical and biochemical experimental studies have made substantial advances and revealed some common features such as cross-β X-ray diffraction patterns. 6,7 Regardless of these advances, detailed information on the structures and formation mechanism of protein aggregates at atomistic levels, and also the interpretation of experimentally revealed structural features based on atomic level studies, are rather limited. For example, we do not yet have clear explanations for the following questions: what molecular features give rise to cross-β spine structure?…”