ATP biphasically modulates LLPS of TDP-43 PLD by specifically binding arginine residues

Abstract

Mysteriously neurons maintain ATP concentrations of ~3 mM but whether ATP modulates TDP-43 LLPS remains completely unexplored. Here we characterized the effect of ATP on LLPS of TDP-43 PLD and seven mutants by DIC and NMR. The results revealed: 1) ATP induces and subsequently dissolves LLPS of TDP-43 PLD by specifically binding Arg saturated at 1:100. 2) ATP modifies the conformation-specific electrostatic property beyond just imposing screening effect. 3) Reversibility of LLPS of TDP-43 PLD and further exaggeration into aggregation appear to be controlled by a delicate network composed of both attractive and inhibitory interactions. Results together establish that ATP might be a universal but specific regulator for most, if not all, R-containing intrinsically-disordered regions by altering physicochemical properties, conformations, dynamics, LLPS and aggregation. Under physiological conditions, TDP-43 is highly bound with ATP and thus inhibited for LLPS, highlighting a central role of ATP in cell physiology, pathology and aging.

Fig. 1. DIC and NMR visualization of ATP in modulating LLPS of TDP-43 PLD.

a Schematic representation of WT and seven mutants of TDP-43 PLD with their pI values indicated. b Turbidity (absorption at 600 nm) of TDP-43 PLD at 15 μM in the presence of ATP at different molar ratios in 10 mM sodium phosphate buffer at pH 5.5 and 7.0, as well as at 7.5 μM at pH 5.5. c DIC images of TDP-43 PLD at 15 μM in the presence of ATP at different molar ratios in the same buffer respective at pH 5.5 and 7.0. d DIC images of TDP-43 PLD at 7.5 μM in the presence of ATP at different molar ratios at pH 5.5. e ¹H-¹⁵N NMR HSQC spectra of the ¹⁵N-labeled TDP-43 PLD at 15 μM in the absence (blue) and in the presence of ATP at a molar ratio of 1:1000 (red) in 10 mM sodium phosphate buffer (pH 5.5). f Chemical shift difference (CSD) of TDP-43 PLD between the free state and in the presence ATP at 1:100 (blue) and 1:1000 (red), respectively. The green line is used to indicate the value: average + STD at the ATP ratio of 1:1000. The residues with CSD values >average + SD are defined as significantly perturbed residues.

Fig. 2. Characterization of the interaction of TDP-43 PLD with NaCl.

a Turbidity of TDP-43 PLD (three measurements) at 15 μM in 10 mM sodium phosphate buffer (pH 5.5) in the presence of NaCl at different concentrations. b HSQC spectra of the ¹⁵N-labeled TDP-43 PLD at 15 μM in the same buffer at pH 5.5 in the free state (blue) and in the presence of NaCl (red) at 50 and 150 mM, respectively. c Chemical shift difference (CSD) of TDP-43 PLD between the free state and in the presence of NaCl at 50 mM (blue) and 150 mM (red). The green line is used to indicate the value: average + SD at 150 mM NaCl.

Fig. 3. DIC and NMR visualization of ATP in modulating LLPS of C1- and C2-PLD.

a Turbidity curves of TDP-43 C1- and C2-PLD at 15 μM in the presence of ATP at different molar ratios in 10 mM sodium phosphate buffer at pH 5.5. b DIC images of TDP-43 C1- and C2-PLD at 15 μM in the presence of ATP at different molar ratios in the same buffer at pH 5.5. c HSQC spectra of the ¹⁵N-labeled TDP-43 C1- and C2-PLD at 15 μM in the same buffer at pH 5.5 in the absence (blue) and in the presence of ATP (red) at a molar ratio of 1:100. d Chemical shift difference (CSD) of TDP-43 C2-PLD between in the absence and in the presence ATP at 1:50 (blue) and 1:100 (red). The green line is used to indicate the value: average + SD at 1:100.

Fig. 4. DIC and NMR visualization of ATP in modulating LLPS of C4- and C6-PLD.

a Turbidity curves of TDP-43 C4- and C6-PLD at 15 μM in the presence of ATP at different molar ratios in 10 mM sodium phosphate buffer at pH 5.5. b DIC images of TDP-43 C4-PLD at 15 μM in the presence of ATP at different molar ratios in the same buffer. c HSQC spectra of the ¹⁵N-labeled TDP-43 C4- and C6-PLD at 15 μM, as well as C5-PLD at 10 μM in the absence (blue) and in the presence of ATP at a molar ratio of 1:1000 (red). d Chemical shift difference (CSD) of TDP-43 C4- (I) and C6-PLD (II) between the free state and in the presence ATP at 1:100 (blue) and 1:1000 (red). The green line is used to indicate the value: average + SD at 1:1000.

Fig. 5. NMR characterization of the interaction of C6-PLD with NaCl.

a Turbidity of TDP-43 C6-PLD (three measurements) at 15 μM in 10 mM sodium phosphate buffer (pH 5.5) in the presence of NaCl at different concentrations. b HSQC spectra of the ¹⁵N-labeled TDP-43 C6-PLD at 15 μM in the absence (blue) and in the presence of NaCl (red) at 50 and 150 mM, respectively. c Chemical shift difference (CSD) of TDP-43 C6-PLD between the free state and in the presence NaCl at 50 mM (blue) and 150 mM (red) (upper) as well as TDP-43 WT-PLD (blue) and C6-PLD (red) in the presence NaCl at 150 mM (lower). The green line is used to indicate the value: average + SD at 150 mM NaCl.

Fig. 6. DIC and NMR visualization of ATP in modulating LLPS of TDP-43 C7-PLD.

a Turbidity curves of TDP-43 C7-PLD at 15 μM and 7.5 μM in the presence of ATP at different molar ratios in 10 mM sodium phosphate buffer at pH 5.5. b DIC images of TDP-43 C7-PLD at 15 μM in the presence of ATP at different molar ratios in the same buffer respective at pH 5.5. c ¹H-¹⁵N NMR HSQC spectra of the ¹⁵N-labeled TDP-43 C7-PLD at 15 μM in the absence (blue) and in the presence of ATP at a molar ratio of 1:500 (red) in 10 mM sodium phosphate buffer (pH 5.5). d Chemical shift difference (CSD) of TDP-43 C7-PLD between the free state and in the presence ATP at 1:100 (blue) and 1:500 (red), respectively. The green line is used to indicate the value (0.05): average + STD at the ATP ratio of 1:500. The residues with CSD values >average + SD are defined as significantly perturbed residues.

Fig. 7. Speculative model for ATP to modulate LLPS of cytoplasmic TDP-43.

a Concentration-dependent CSD for the significantly perturbed residues of TDP-43 WT-, C4-, C6-, and C7-PLD. b The speculative mechanism for ATP to biphasically modulate LLPS of RGG-rich FUS CTD. c The speculative mechanism for ATP to biphasically modulate LLPS of TDP-43 PLD. d A speculative model for ATP to modulate LLPS of cytoplasmic TDP-43 and its pathological implications. (I) Under the physiological conditions, LLPS is inhibited due to being bound with ATP of cytoplasmic TDP-43 in neurons, where TDP-43 has concentrations of ~1 μM while ATP has concentrations of ~3 mM. (II) Cytoplasmic TDP-43 reversibly phase separates into dynamic droplets which might be enhanced by the bivalent binding of ATP under pathological/aging conditions with the accumulation of TDP-43, or/and reduction of ATP concentrations. (III) Cytoplasmic TDP-43 droplets become capable of recruiting other proteins. (IV) With a long incubation time, TDP-43 droplets may undergo an irreversible exaggeration from the dynamic droplets into aggregates or/and amyloid fibrils characteristic of ALS.