A blueprint for functional engineering: Single point mutations reconstitute phosphatidylinositol presentation in a...

Phosphoinositides, phosphorylated species of phosphatidylinositol (PtdIns), are critical regulatory lipids in all eukaryotic cells. The molecular mechanisms that lead to the phosphorylation of an individual PtdIns- or phosphoinositide molecule remain largely unkown even though lipid kinases and phosphatases involved in these processes have been studied in detail. The observation by us and others that liposomal PtdIns (and phosphoinositide) molecules are poor in vitro substrates for kinases and phosphatases raises the question of how these enzymes execute their function in living cells. Recent work indicates that Sec14, the founding member of a large superfamily of eukaryotic proteins, is crucial for the process of PtdIns phosphorylation. The collective data suggest that Sec14 mediates a heterotypic phospholipid exchange reaction of PtdIns with phosphatidylcholine (PtdCho) during which PtdIns becomes vulnerable for kinase attack and thereby promotes the generation of phosphoinositides.1,2 In a recent paper we address the molecular mechanism of this phospholipid (PL) exchange reaction in a pseudo-Sec14 protein (Sfh1) that we rendered functional by a directed evolution approach. We find that enhanced PL-cycling into and out of the hydrophobic pocket of these activated Sfh1 mutants depends on the reconfiguration of interactions between a C-terminal string motif and the floor of the hydrophobic pocket that results in increased oscillations in a helical gate that controls pocket access. Here we further discuss our findings and propose molecular dynamics simulations as a tool to approach energetically unfavorable transition states and to identify novel protein-ligand interactions invisible to X-ray crystallography.