ATP regulates TDP-43 pathogenesis by specifically binding to an inhibitory component of a delicate network controlling LLPS
ABSTRACTVery recently, liquid-liquid phase separation (LLPS) of cytoplasmic TDP-43 independent of forming stress granule (SG) has been decoded to initiate the neuron death for ALS. Mysteriously neurons maintain ATP concentrations of ∼3 mM, but whether ATP modulates TDP-43 LLPS remains completely unknown. Here we characterized the effects of ATP on LLPS of TDP-43 PLD and its six mutants by DIC and NMR. For the first time, the results revealed: 1) ATP does induce and subsequently dissolve LLPS of TDP-43 PLD. 2) ATP achieves the modulation all by specifically binding Arg mostly saturated at 1:100. 3) LLPS of TDP-43 PLD and its exaggeration into aggregation are controlled by a delicate network composed of both attractive and inhibitory interactions, thus rationalizing the susceptibility of TDP-43 PLD to various ALS-causing mutations. Our studies together establish that ATP specifically binds Arg in intrinsically-disordered domains even not RGG-/R-rich, implying that ATP might be a universal regulator for most, if not all, R-containing intrinsically-disordered domains by altering their physicochemical features, conformations, dynamics, LLPS and assembly. Under physiological conditions, TDP-43 even in neuronal cytoplasm is highly bound with ATP and thus inhibited for its toxic LLPS, highlighting a central role of ATP in TDP-43 pathogenesis and aging.