Inositol Pyrophosphate Specificity of the SPX-Dependent Polyphosphate Polymerase VTC

The free energy of nucleotide hydrolysis depends on phosphate concentration. Cells regulate cytosolic phosphate levels by orchestrating phosphate acquisition and storage through inositol pyrophosphates (PP-InsP) and SPX domains. Here, we report the synthesis of the novel 5-PPP-InsP₅ containing a triphosphate subunit. Using this and a series of synthetic PP-InsP, we examined the ligand specificity of the SPX domain in the PP-InsP-controlled yeast polyphosphate polymerase VTC. SPX decodes the relative positioning of the phosphoric anhydrides, their structure (diphosphate vs triphosphate), and the presence of other phosphates on the inositol ring. Despite the higher potency of 1,5-(PP)₂-InsP₄, 5-PP-InsP₅ is the primary activator of VTC in cells, indicating that its higher concentration compensates for its lower potency. 1,5-(PP)₂-InsP₄ levels rise and could become relevant under stress conditions. Thus, SPX domains may integrate PP-InsP dependent signaling to adapt cytosolic phosphate concentrations to different metabolic situations.