Mechanistic Insight on General Protein-Binding Ability of ATP and the Impacts of Arginine Residues

Abstract

Recent experiments suggested that adenosine triphosphate (ATP) can regulate liquid–liquid phase separation (LLPS) of various proteins and inhibit protein aggregations at its physiological concentration, which is highly correlated with the nonspecific interactions of ATP to a wide variety of proteins. However, the mechanism underlying the general binding capability of ATP largely remains unclear. In this work, we used molecular dynamics simulation to study the binding of ATPs to three proteins with distinct net charges: TDP-43 NTD (−7 e), TAF15-RRM (0 e), HWEL (+8 e). Negatively charged ATP exhibits a strong trend to accumulate around all of these proteins. While only a fraction of the accumulated ATPs directly binds to the limited regions of the protein surface, additional ATPs indirectly bind to proteins by aggregating into ATP clusters. Hence, the proportion of the directly bound ATPs in the clusters as well as their binding regions can be adjusted in response to different proteins, which makes ATP well adapted to a variety of proteins. Moreover, our results suggest that ATP tightly binds to Arg with high affinity, and Arg dominates the direct binding of ATP. Meanwhile, Arg also affects the self-association of accumulated ATPs. The size of the ATP cluster is effectively regulated by the distribution of Arg. Considering the ubiquity of Arg in proteins, our findings are helpful to understand the general binding capability of ATP.

Nonspecific Binding of Adenosine Tripolyphosphate and Tripolyphosphate Modulates the Phase Behavior of Lysozyme

“…This data clearly shows that both ATP and TPP preferentially bind to arginine over lysine residues. This is in line with recent reports that ATP preferentially interacts with arginine residues on the protein surfaces, and Arg to Lys mutations impede these interactions. ,, …”

“…An important result of our study is that ATP and TPP preferentially bind to arginine groups. This finding is supported by molecular simulations of lysozyme and closely follows the polyphosphate ion binding propensity for IDR regions. ,,, Given that ATP strongly interacts with arginine groups irrespective of protein disorder or flexibility, there may also be similarities in how ATP modifies solubility of IDPs compared to folded proteins. The increased insolubility and phase separation tendency of IDPs has been attributed in part due to the stickiness of arginine, which arises from the ability to form cation−π and π–π interactions most commonly with aromatic groups.…”

“…An important result of our study is that ATP and TPP preferentially bind to arginine groups. This finding is supported by molecular simulations of lysozyme 49 and closely follows the polyphosphate ion binding propensity for IDR regions. 17,25,26,29 Given that ATP strongly interacts with arginine groups irrespective of protein disorder or flexibility, there may also be similarities in how ATP modifies solubility of IDPs compared to folded proteins.…”

“…This is in line with recent reports that ATP preferentially interacts with arginine residues on the protein surfaces, and Arg to Lys mutations impede these interactions. 25,29,49 ATP Binding to Individual Binding Sites Is Not Affected by the Presence of Mg 2+ Ions. Using 31 P NMR, we established that a fully saturated ATP−Mg complex forms at equimolar concentrations of ATP and MgCl 2 (Figure S8), with Mg 2+ predominantly complexed between β and γ phosphate groups of ATP, which is in line with previous reports.…”

Distinct conformational changes occur within the intrinsically unstructured pro‐domain of pro‐Nerve Growth Factor in the presence of ATP and Mg2+

“…Overall, our experiments show that under thermodynamic equilibrium conditions, (i) the ATP/Mg 2+ and ATP/rh‐proNGF moieties compete, (ii) that a ternary rh‐proNGF/ATP/Mg 2+ complex does not form, and (iii) that ATP binding to rh‐proNGF can be modulated by the relative concentration of the three components (rh‐proNGF, ATP and Mg 2+ ). This suggests an interplay of an unspecific electrostatic component to ATP/rh‐proNGF binding and a likely direct interaction—a very specific one—of ATPs negatively charged phosphate groups with positively charged protein residues (arginines, lysines, and histidines) (Hu et al, 2022 ). Interestingly, the pro‐peptide domain of rh‐proNGF is characterized by a higher proportion of positively charged residues, compared to the mature NGF domain (Figure 5a ).…”