Investigating the Interaction Mechanism and Effect of ATP on Alpha-Synuclein Aggregation by NMR Spectroscopy

Abstract

Recent studies suggest that Adenosine Triphosphate (ATP) can either enhance or inhibit the aggregation of amyloid proteins, depending on the interaction mechanism as well as specific protein properties. The connection between ATP and protein solubility is particularly important in Parkinson’s Disease (PD), where the aggregation of alpha-synuclein (αS) is closely linked to pathology. Since the greatest risk factor for PD is aging, and ATP levels decline dramatically with age and are greatly reduced in the brains of patients with early PD, it is possible that the modulating effect of ATP on protein solubility is a factor in PD onset. However, the driving mechanism behind the interaction of ATP and αS is currently unclear, as is the effect of physiologically-relevant ATP concentrations on early- and late-stage αS aggregation. Here, we determine using NMR spectroscopy that the triphosphate moeity of ATP drives its electrostatic interaction primarily with the N-terminal pseudo-apolipoprotein repeats of αS monomers. These interactions are modulated by magnesium and disrupt long-range N- to C-terminal contacts in αS monomers, causing a concentration-dependent enhancement of initial αS aggregation. We also show by Thioflavin T fluorescence as well as electron microscopy that ATP inhibits late-stage αS β-sheet fibril formation in a phosphate-dependent manner. Our NMR data reveals that ATP inhibits αS monomer-fibril interactions, suggesting that ATP attenuates αS secondary nucleation. Lastly, we show that the effects of ATP are different in the presence of PD-related αS mutations E46K and A53T. Overall, our study contributes a thorough characterization of the biologically- and pathologically-relevant interactions between ATP and αS, while also proposing a role for ATP in the age-related development of PD pathology.