The crystal structure of the phosphatidylinositol 4-kinase IIα

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4‐kinase type IIα (PI4K IIα), in complex with ATP solved by X‐ray crystallography at 2.8 Å resolution. The structure revealed a non‐typical kinase fold that could be divided into N‐ and C‐lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C‐lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane. Synopsis The crystal structure of PI4K IIα reveals its membrane binding mode and highlights a membrane‐oriented hydrophobic pocket as a potential allosteric regulatory site. The crystal structure of PI4K IIα was solved at 2.8 Å resolution. The structure highlights important differences between type II and type III PI 4‐kinases. Molecular simulation suggests a membrane binding mode that involves a hydrophobic pocket with allosteric regulatory potential. Graphical Abstract The crystal structure of PI4K IIα reveals its membrane binding mode and highlights a membrane‐oriented hydrophobic pocket as a potential allosteric regulatory site.