Cellular ATP Levels Determine the Stability of a Nucleotide Kinase

Abstract

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

Disease-Related Protein Variants of the Highly Conserved Enzyme PAPSS2 Show Marginal Stability and Aggregation in Cells

“…Missense mutations in nuclear deubiquitinase BAP1 were previously shown to induce destabilization and aggregation of this enzyme, with the latter being suggested as the main cause of its functional loss (Bhattacharya et al, 2016). In fact, this hypothesis is further supported by our previous studies (Brylski et al, 2021) using an alanine scanning mutagenesis of the substrate binding site of APSK37 in HeLa cells. The results revealed a large range of different in-cell stabilities for the single point mutations (ΔG f 0 = −10.7 to +13.8 kJ/mol).…”

“…We analyzed the effect of disease-related mutations on the folding stability and aggregation of the APS kinase domain of the bifunctional PAPSS2 protein, using our recently established APSK folding sensor (APSK37) (Brylski et al, 2021). The sensor reports intramolecular FRET between the N-terminal AcGFP1 and the C-terminal mCherry fusion proteins (see Materials and Methods for details).…”

“…Using a recently developed folding sensor of the APS kinase domain of the human PAPS synthase PAPSS2 (Brylski et al, 2021), we investigate how clinically reported single-pointmutations change the in-cell stability of the APS kinase domain and if destabilization could lead to aggregation and thus loss of metabolic activity.…”

CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute

“…The function of ATP acting as a cosolute to maintain biomolecular processes in cells could explain why the cellular ATP concentration (5–10 mM) exceeds the level required to sustain energy-consuming processes by a factor of up to 1000. ,,, In line with this hypothesis, previous studies showed that elevated ATP levels could increase protein solubility and maintain the liquid-like properties of nucleoli, − stress granules, , and, in general, the cytoplasm of eukaryotes and prokaryotes. ,,,, …”

“…To probe whether changes in the ATP concentration also affect the folding stability of (CAG) 20 and lm4U* in cells, we conducted ATP depletion experiments, adding final concentrations of 1 mM KCN and 10 mM 2-deoxyglucose to the medium . ATP depletion was monitored by the ATeam ATP concentration sensor (see Figure S9 and Supporting Information Materials and Methods for further details), , and (CAG) 20 RNA folding stability was assessed at the minimum ATP level. The normalized FRET ratio decreased from 1.0 to 0.6, corresponding to a decrease in the cellular ATP concentration by 2–3 mM .…”