ATP energy-independently modulates the folding equilibrium of ALS-causing C71G-hPFN1

Abstract C71G is the most aggregation-prone and toxic mutant of 140-residue human profin-1 (hPFN1) that causes familial ALS by gain of toxicity, but its underlying mechanisms still remain unknown. C71G-hPFN1 exists in an equilibrium between folded and unfolded states, whose dynamic/thermodynamic properties and modulation are not yet defined. Here, we utilized NMR to quantify the populations to be 55.2% and 44.8% respectively for folded and unfolded states exchanging at 11.7 Hz. Intriguingly, even the folded state of C71G-hPFN1 has increased ps-ns flexibility and reduced thermodynamic stability, thus rationalizing its high aggregation-proneness. Strikingly, C71G-hPFN1 provides a unique model to unambiguously visualize the effects of ATP and 11 related molecules on its folding equilibrium by NMR. Unexpectedly, ATP completely converted C71G-hPFN1 into the folded state at 1:2, which is physiologically relevant in most living cells. By contrast, TMAO, a well-known protein-folding inducer, showed no detectable conversion even at 1:2000. Surprisingly, the inducing capacity of ATP comes from its triphosphate group, but free triphosphate strongly triggered aggregation. The inducing capacity was determined to rank as: ATP = ATPP = PPP > ADP = AMP-PNP = AMP-PCP = PP, while AMP, Adenosine, P and NaCl showed no detectable capacity. Mechanistically, ATP and triphosphate appear to enhance the intrinsic folding capacity encoded by the sequence. Therefore, by joining Adenosine and triphosphate ATP appears to integrate three abilities: inducing folding, inhibiting aggregation and increasing stability. Our study provide a mechanism for the finding that some single-cell organisms still use polyphosphates as primordial chaperones. Moreover, ATP continue to play foundational roles in modern cells, shedding light on the longstanding enigma of the age-related onset of FALS, which coincides with the ageing-dependent reductions in ATP concentrations.