The activation loop of PIP5K functions as a membrane sensor essential for processing of lipid substrates

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), a representative member in the phosphatidylinositol phosphate kinase (PIPK) family, is the major enzyme biosynthesizing the signaling molecule PI(4,5)P 2 in eukaryotic cells. The stringent specificity towards lipid substrates and the high sensitivity to membrane environment strongly suggest a membrane sensing mechanism, but the underlying structural basis is still largely unknown. Here, we present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K's activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with 1,2‐dihexanoyl‐ sn ‐glycero‐3‐phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane sensing mechanism of PIP5K. The activation loop surprisingly functioning as a membrane sensor represents a new paradigm of kinase regulation by activation loop through protein‐membrane interaction, which also lays a foundation for future study on the regulation of PIP5K (and other PIPKs) by membrane lipids. Support or Funding Information This work is supported by Michigan State University Start‐up Fund.Proposed activation mechanism of PIP5K.