A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein.

Αlpha-synuclein (αS) is an intrinsically disordered protein which exhibits a high degree of conformational heterogeneity. In vivo, αS experiences various environments which cause adaptation of its structural ensemble. Divalent metal ions are prominent in synaptic terminals where αS is located and are thought to bind to the αS C-terminal region. Herein, we used native nanoelectrospray ionization ion mobility-mass spectrometry to investigate changes in the charge state distribution and collision cross sections of wild-type N-terminally acetylated (NTA) αS, along with a deletion variant (ΔΔNTA) which inhibits amyloid formation and a C-terminal truncated variant (119NTA) which increases the rate of amyloid formation. We also examine the effect of the addition of divalent metal ions, Ca2+, Mn2+, and Zn2+, and correlate the conformational properties of the αS monomer with the ability to aggregate into amyloid, measured using Thioflavin T fluorescence and negative stain transmission electron microscopy. We find a correlation between the population of species with a low collision cross section and accelerated amyloid assembly kinetics, with the presence of metal ions resulting in protein compaction and causing ΔΔ to regain its ability to form an amyloid. The results portray how the αS conformational ensemble is governed by specific intramolecular interactions that influence its amyloidogenic behavior.

Single residue modulators of amyloid formation in the N-terminal P1-region of α-synuclein.

Alpha-synuclein (αSyn) is a protein involved in neurodegenerative disorders including Parkinson's disease. Amyloid formation of αSyn can be modulated by the 'P1 region' (residues 36-42). Here, mutational studies of P1 reveal that Y39A and S42A extend the lag-phase of αSyn amyloid formation in vitro and rescue amyloid-associated cytotoxicity in C. elegans. Additionally, L38I αSyn forms amyloid fibrils more rapidly than WT, L38A has no effect, but L38M does not form amyloid fibrils in vitro and protects from proteotoxicity. Swapping the sequence of the two residues that differ in the P1 region of the paralogue γSyn to those of αSyn did not enhance fibril formation for γSyn. Peptide binding experiments using NMR showed that P1 synergises with residues in the NAC and C-terminal regions to initiate aggregation. The remarkable specificity of the interactions that control αSyn amyloid formation, identifies this region as a potential target for therapeutics, despite their weak and transient nature.