Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution.

Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.

A blueprint for functional engineering: Single point mutations reconstitute phosphatidylinositol presentation in a pseudo-Sec14 protein.

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.