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Cellular sulfation pathways rely on the activated sulfate 3′-phosphoadenosine-5′-phosphosulfate (PAPS). In humans, PAPS is exclusively provided by the two PAPS synthases PAPSS1 and PAPSS2. Mutations found in the PAPSS2 gene result in severe disease states such as bone dysplasia, androgen excess and polycystic ovary syndrome. The APS kinase domain of PAPSS2 catalyzes the rate-limiting step in PAPS biosynthesis. In this study, we show that clinically described disease mutations located in the naturally fragile APS kinase domain are associated either with its destabilization and aggregation or its deactivation. Our findings provide novel insights into possible molecular mechanisms that could give rise to disease phenotypes associated with sulfation pathway genes.

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.

Within the crowded and complex environment of the cell, a protein experiences stabilizing excluded-volume effects and destabilizing quinary interactions with other proteins. Which of these prevail, needs to be determined on a case-by-case basis. PAPS synthases are dimeric and bifunctional enzymes, providing activated sulfate in the form of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) for sulfation reactions. The human PAPS synthases PAPSS1 and PAPSS2 differ significantly in their protein stability as PAPSS2 is a naturally fragile protein. PAPS synthases bind a series of nucleotide ligands and some of them markedly stabilize these proteins. PAPS synthases are of biomedical relevance as destabilizing point mutations give rise to several pathologies. Genetic defects in PAPSS2 have been linked to bone and cartilage malformations as well as a steroid sulfation defect. All this makes PAPS synthases ideal to study protein unfolding, ligand binding, and the stabilizing and destabilizing factors in their cellular environment. This review provides an overview on current concepts of protein folding and stability and links this with our current understanding of the different disease mechanisms of PAPSS2-related pathologies with perspectives for future research and application.