Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2 with positive feedback

The ability for cells to localize and activate peripheral membrane binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes in eukaryotic cells are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyze the phosphorylation of PI(4)P to generate most PI(4,5)P2 lipids found in cells. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. Here, we examine how two PIP5K motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PIP lipids. Attaching the PIP5K specificity loop to other peripheral membrane binding proteins can enhance their membrane binding dynamics. After correctly orienting on the membrane, PIP5K transitions to binding PIP lipids in a structural motif previously referred to as the substrate or PIP binding motif (PIPBM). Our data reveals that the PIPBM has broad specificity for anionic lipids and serves a critical role in regulating membrane association in vitro and in vivo. The strength of the interaction between the PIPBM and PIP lipids depends on the membrane density and the extent phosphorylation on the inositol head group. We propose a two-step membrane binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production.