Cellular ATP Levels Determine the Stability of a Nucleotide Kinase.

Oliver Brylski,Puja Shrestha,Simon Ebbinghaus,Patricia Gnutt,Jonathan Wolf Mueller,David Gnutt

doi:10.3389/fmolb.2021.790304

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 ΔGf 0 = -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.

Highlights

In eukaryotic cells, there is abundant ATP at millimolar concentrations (Traut, 1994)
For an enzyme like PAPS synthase where all stabilizing substrates need to be generated from ATP, stability of the enzyme is necessarily connected to the availability of ATP inside the cell
Using alanine-mutagenesis and ATP depletion experiments of APSK37 in living cells, we investigated the effect of ligand binding on APSK37 stability and how it is connected to cellular ATP levels

Summary

Introduction

There is abundant ATP at millimolar concentrations (Traut, 1994). Under conditions of stress, such as starvation (Maddocks et al, 2013; Petrovska et al, 2014) or DNA damage (Bonora et al, 2012), the ATP concentration is fluctuating inside cells. A class of proteins traditionally linked to ATP are P-loop kinases; they are ATP-dependent phosphate-transferring enzymes (Leipe et al, 2003). A special class of nucleotide kinases are those that phosphorylate the atypical nucleotide adenosine-5′-phosphosulfate (APS). Because APS is created in an upstream reaction that strongly relies on ATP as a substrate (Mueller and Shafqat, 2013), all substrates and all products of the reaction catalyzed by APS kinase (APSK) directly or indirectly depend on ATP availability (Brylski et al, 2019)

Methods

Results

Conclusion