Uncovering the molecular mechanism for dual effect of ATP on phase separation in FUS solution

Abstract

Recent studies reported that adenosine triphosphate (ATP) could inhibit and enhance the phase separation in prion-like proteins. The molecular mechanism underlying such a puzzling phenomenon remains elusive. Here, taking the fused in sarcoma (FUS) solution as an example, we comprehensively reveal the underlying mechanism by which ATP regulates phase separation by combining the semiempirical quantum mechanical method, mean-field theory, and molecular simulation. At the microscopic level, ATP acts as a bivalent or trivalent binder; at the macroscopic level, the reentrant phase separation occurs in dilute FUS solutions, resulting from the ATP concentration–dependent binding ability under different conditions. The ATP concentration for dissolving the protein condensates is about 10 mM, agreeing with experimental results. Furthermore, from a dynamic point of view, the effect of ATP on phase separation is also nonmonotonic. This work provides a clear physical description of the microscopic interaction and macroscopic phase diagram of the ATP-modulated phase separation.

ATP and nucleic acids competitively modulate LLPS of the SARS-CoV2 nucleocapsid protein

“…By contrast, for the nucleic-acid-binding domains 30 such as FUS RRM domain 29 , 33 and NTD/CTD of SARS-CoV-2 N protein, ATP specifically binds to the pockets within their nucleic-acid-binding interface, which is formed only upon the correct folding by diverse residues located over the entire sequence. On the other hand, for IDRs ATP and nucleic acid were both shown to bind Arg/Lys residues in the residue-specific manner by establishing electrostatic interactions between triphosphate group pf ATP or phosphate group of nucleic acid and side chain cations of Arg/Lys as well as π-π/π-cation interactions between base aromatic rings and Arg/Lys side chains, as we experimentally found on FUS 28 and TDP-43 PLD 35 , 41 as well as very recently uncovered by combining quantum mechanical method, mean-field theory and molecular simulations 63 .…”

Arg/Lys-containing IDRs are cryptic binding domains for ATP and nucleic acids that interplay to modulate LLPS

“…Briefly, different from the binding to a well-folded protein to form the stable classic complex with a well-defined three-dimensional structure in which various types of residues are involved and a single atom variation of ATP/nucleic acids or proteins might dramatically alter the binding affinity, in forming the dynamic complex with IDRs which lack the defined conformation and thus are highly accessible to the bulk solvent, ATP/nucleic acids can only establish the NMR-detectable binding to Arg/Lys residues through electrostatic interactions between phosphate groups of ATP/nucleic acids and side chain cations of Arg/Lys as well as π–π/π–cation interactions between base aromatic rings and Arg/Lys side chains in which different bases have a highly similar affinity to Arg/Lys residues. Very recently, such interactions of ATP to Arg have also been uncovered by combining the semiempirical quantum mechanical method, mean-field theory, and molecular simulations 47 , which are fundamentally different from the non-specific electrostatic/salt effects and thus provide the specific capacity to ATP/nucleic acids for driving LLPS of Arg/Lys-containing IDRs. In this context, RNA and ssDNA are expected to bind Arg/Lys residues of IDRs with the same mechanism, because they only have two minor differences in their chemical structures.…”

“…thus resulting in different binding modes and excluding volumes. Chemically, ATP has a unique triphosphate group which appears to have a strong capacity in interacting with water molecules [35][36][37][38][39] and the Arg side chain 47 , These differences may affect the competition between ATP and nucleic acid in binding Arg/Lys residues as well as their abilities to modulate LLPS. Intriguingly, although nucleic acid may acquire very high binding affinity to IDR by establishing multivalent but discrete binding to multiple Arg/Lys residues, such a high-affinity binding appears to be relatively vulnerable to be displaced by ATP as each of the discrete binding events can be simultaneously displaced by ATP.…”

Polyanion order controls liquid-to-solid phase transition in peptide/nucleic acid co-assembly

“…Varying ATP concentrations in cells can work to control protein condensation, ( Patel et al, 2017 ; Aida et al, 2022 ; Saurabh et al, 2022 ) and biphasic control of PLD proteins like FUS in membraneless compartments relies on electrostatic and π-cation interactions that, in a concentration-dependent manner, both condenses and dissolves protein condensates ( Patel et al, 2017 ; Ren et al, 2022 ). Phosphate salts are also strongly kosmotropic and are known to induce crystalline order in other biomolecular condensates ( Malay et al, 2020 ).…”

“…The multifaceted functions that RNP granules play depends on dynamic structural plasticity of these co-assemblies ( Banani et al, 2017 ; Boeynaems et al, 2019 ; Conicella et al, 2020 ; Dutagaci et al, 2021 ; Gordon et al, 2021 ; Hallegger et al, 2021 ; Carey and Guo, 2022 ). For example, the cooperative interactions between arginine residues of the RNA binding domain and the tyrosine residues from the prion-like domains (PLD) of FUS are critical for the dual effects of ATP concentrations that both induces and inhibits initial phase transitions at different concentrations by controlling these interactions ( Wang et al, 2018 ; Ren et al, 2022 ).…”