ATP induces folding of ALS-causing C71G-hPFN1 and nascent hSOD1

Protein folding is crucial for function, but misfolding and aggregation are hallmarks of aging and neurodegenerative diseases. ATP, the cellular energy currency, is present at high concentrations exceeding its known functional needs. Recent research suggests ATP's involvement in protein hemostasis, dissolving aggregates and influencing liquid-liquid phase separation. Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with familial (FALS) and sporadic (SALS) forms. Mutations in human CuZn-superoxide dismutase 1 (hSOD1) cause FALS, and hSOD1 misfolding is implicated in SALS. Nascent hSOD1 is unfolded, while its mature form is a stable homodimer. Mutations in human profilin 1 (hPFN1), such as C71G, also cause FALS, with C71G-hPFN1 existing in a dynamic equilibrium between folded and unfolded states. This study investigates the effect of ATP on the folding equilibrium of C71G-hPFN1 and nascent hSOD1 to understand ALS pathogenesis.

Extensive research has focused on the mechanisms of protein folding and the role of chaperones in preventing misfolding and aggregation. The high cellular concentrations of ATP have prompted investigations into its non-energetic roles in protein homeostasis. Studies have shown ATP's ability to dissolve protein aggregates, modulate liquid-liquid phase separation of intrinsically disordered proteins, and inhibit amyloid fibrillation. However, the direct effect of ATP on protein folding equilibrium remained largely unexplored. The involvement of hSOD1 and hPFN1 mutations in ALS has been widely studied, but the precise mechanisms leading to misfolding and aggregation are still unclear. NMR studies have characterized the unfolded nature of nascent hSOD1 and the equilibrium between folded and unfolded states of C71G-hPFN1. Prior research established the role of zinc ions in initiating the folding of nascent hSOD1 and the link between misfolding and toxicity of C71G-hPFN1.

The study used NMR spectroscopy to characterize the conformational equilibrium and dynamics of WT-hPFN1 and C71G-hPFN1. Sequential assignments were made using triple-resonance NMR spectra. 15N backbone relaxation data (R1, R2, and hNOE) provided information on ps-ns backbone dynamics. 3D heteronuclear HSQC-NOESY spectra were used to quantify the populations and exchange parameters of the folding equilibrium of C71G-hPFN1. Pulsed field gradient NMR self-diffusion measurements determined the translational diffusion coefficients. NMR titrations with ATP and various related molecules were performed to assess their effects on the folding equilibrium of C71G-hPFN1. Similar NMR experiments were conducted for nascent hSOD1, including titrations with Zn2+ and ATP-related molecules. The thermodynamic stability of C71G-hPFN1 and WT-hPFN1 was measured using differential scanning fluorimetry (DSF). The solution conformations and dynamics of ATP- and Zn2+-induced folded states of hSOD1 were compared by superimposing HSQC spectra, collecting triple-resonance NMR spectra, and analyzing hNOE data. Titrations with TMAO were performed to compare its effect with ATP.

NMR analysis revealed that C71G-hPFN1 exists in a dynamic equilibrium between folded and unfolded states, with approximately 55.2% in the folded state and 44.8% in the unfolded state, exchanging at a rate of ~11.7 Hz. The folded state of C71G-hPFN1 showed increased backbone flexibility compared to WT-hPFN1. ATP, at a 1:2 molar ratio, completely converted C71G-hPFN1 to the folded state. The triphosphate moiety of ATP was identified as the key component responsible for inducing folding. However, free triphosphate also triggered aggregation. The capacity to induce folding was ranked as: ATP > ATPγS > ADP > AMP-PNP = AMP-PCP > PP, while AMP, adenosine, Pi, and NaCl showed no effect. ATP induced the folding of nascent hSOD1, but only at a 1:8 molar ratio, resulting in a mixture of folded and unfolded states. The mechanism of ATP-induced folding differed from that of Zn2+-induced folding. TMAO, a known protein folding inducer, failed to induce the folding of either C71G-hPFN1 or nascent hSOD1, even at high concentrations. ATP enhanced the thermodynamic stability of C71G-hPFN1 but not WT-hPFN1. Comparative analysis of ATP- and Zn2+-induced folded states of hSOD1 showed that ATP mainly influences the loop regions.

This study provides a detailed mechanistic understanding of how ATP induces folding of ALS-causing proteins. The triphosphate group's ability to interact with and displace water molecules from the protein backbone appears to be the driving force behind this effect. The adenosine moiety likely plays a critical role in preventing aggregation by interacting with hydrophobic patches. The findings support the idea that polyphosphates acted as primordial chaperones, and suggest that ATP continues to play a vital, energy-independent role in modern cells, preventing protein misfolding and aggregation. The age-related onset of familial ALS may be related to the age-dependent decrease in cellular ATP concentration. The different mechanisms of ATP and Zn2+ induced folding highlight the complexity of protein folding.

This study demonstrates ATP's ability to induce folding of ALS-related proteins C71G-hPFN1 and nascent hSOD1. The triphosphate moiety is crucial for this effect, but the adenosine moiety plays a role in preventing aggregation. The findings provide insight into the role of ATP in protein homeostasis, suggesting a potential therapeutic strategy for ALS. Future studies should explore the energy-independent functions of ATP in more complex cellular environments.

The study primarily focused on in vitro experiments using purified proteins. The results may not fully reflect the complexities of the cellular environment, where macromolecular crowding and other factors can influence protein folding. The sample preparation and buffer conditions were optimized to minimize aggregation, potentially limiting the generalizability of some findings to physiological conditions.