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Threonine Cavities Are Targetable Motifs That Control Alpha-Synuclein Fibril Growth
Noah Nathan Kochen1, Vivek Vasandani1, Darren Seaney1
1Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Abstract:
Recent high-resolution structures of alpha-synuclein (aSyn) fibrils offer promise for rational approaches to drug discovery for Parkinson's disease and Lewy body dementia. Harnessing the first such structures, we previously used molecular dynamics and free energy calculations to suggest that threonines 72 and 75─which line water-filled cavities within the fibril stacks─may be of central importance in stabilizing fibrils. Here, we used experimental mutagenesis of both wild-type and A53T aSyn to show that both threonine residues play important but surprisingly disparate roles in fibril nucleation and elongation. The T72A mutant, but not T75A, resulted in a large increase in the extent of fibrillization during primary nucleation, leading us to posit that T72 acts as a "brake" on run-away aggregation. An expanded set of simulations of five recent high-resolution fibril structures suggests that confinement of cavity waters around T72 correlates with this finding. In contrast, the T75A mutation led to a modest decrease in the extent of fibrillization. Furthermore, both T72A and T75A completely blocked the initial fibril elongation in seeded fibrillization. To test whether these threonine-lined cavities are druggable targets, we used computational docking to identify potential small-molecule binders. We show that the top-scoring hit, aprepitant, strongly promotes fibril growth while specifically interacting with aSyn fibrils and not monomer, and we offer speculation as to how such compounds could be used therapeutically.
Insights
Threonine residues in alpha-synuclein fibrils play critical roles in Parkinson's disease aggregation. T72 acts as a brake on aggregation, while T75 has a lesser role, and both block elongation, suggesting druggable targets.
Area of Science:
- Neuroscience
- Biochemistry
- Structural Biology
Background:
- High-resolution structures of alpha-synuclein (aSyn) fibrils are crucial for understanding Parkinson's disease (PD) and Lewy body dementia (LBD).
- Previous studies suggested threonine residues 72 and 75 (T72, T75) within aSyn fibrils are key to fibril stabilization.
- These residues line water-filled cavities within fibril stacks.
Purpose of the Study:
- To investigate the specific roles of T72 and T75 in alpha-synuclein fibril formation using experimental mutagenesis.
- To explore the druggability of threonine-lined cavities in aSyn fibrils for potential therapeutic interventions.
Main Methods:
- Site-directed mutagenesis of wild-type and A53T alpha-synuclein (aSyn) to create T72A and T75A mutants.
- Fibrillization assays to assess primary nucleation and elongation.
- Molecular dynamics simulations of multiple high-resolution aSyn fibril structures.
- Computational docking to identify small-molecule binders targeting aSyn fibrils.
Main Results:
- T72A mutation significantly increased fibrillization during primary nucleation, suggesting T72 acts as an aggregation brake.
- T75A mutation resulted in a modest decrease in fibrillization extent.
- Both T72A and T75A mutations completely inhibited initial fibril elongation in seeded assays.
- Confinement of cavity waters around T72 correlated with increased aggregation in simulations.
- Aprepitant, a small molecule, was identified as a binder that promotes fibril growth by interacting with aSyn fibrils.
Conclusions:
- Threonine residues T72 and T75 have distinct and significant roles in alpha-synuclein fibril formation, impacting both nucleation and elongation.
- The threonine-lined cavities within aSyn fibrils represent potential druggable targets.
- Compounds like aprepitant that modulate aSyn fibril formation could offer novel therapeutic strategies for PD and LBD.
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