Disulfide engineering reveals unexpected pro- and anti-aggregation conformers of human α-synuclein

Area of Science:

• Biochemistry
• Structural Biology
• Neuroscience

Background:

• Intrinsically disordered proteins (IDPs) exhibit conformational heterogeneity, forming distinct polymorphs.
• Alpha-synuclein (α-synuclein) aggregation into amyloid fibrils is central to Parkinson's disease.
• Disulfide crosslinks can stabilize specific protein conformations.

Purpose of the Study:

• To investigate the role of intramolecular disulfide bonds in controlling α-synuclein aggregation.
• To explore the formation of distinct α-synuclein amyloid polymorphs using engineered crosslinks.

Main Methods:

• Creation of six double-cysteine α-synuclein variants.
• Purification of disulfide-crosslinked α-synuclein monomers.
• Analysis of amyloid fibril formation using negative-stain transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM).

Main Results:

• Two predicted pro-aggregation variants formed expected amyloid fibrils.
• One control variant unexpectedly formed amyloid fibrils with a novel morphology.
• A crosslinked variant that failed to aggregate exhibited potent anti-aggregation activity against wild-type α-synuclein.

Conclusions:

• Engineered disulfide crosslinks can direct α-synuclein into specific aggregation pathways, potentially forming new polymorphs.
• Disulfide crosslinking offers a strategy to control α-synuclein aggregation and explore its conformational landscape.
• One crosslinked variant demonstrates potential as a therapeutic agent against α-synuclein aggregation.