Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution
MetadataShow full item record
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. © 2011 Schaaf et al.
Showing items related by title, author, creator and subject.
Falasca, Marco; Maffucci, T. (2009)A generally accepted view considers phosphatidylinositol 3-monophosphate (PtdIns3P) as a lipid confined to the endosomal compartment where it regulates trafficking pathways and is produced constitutively and exclusively ...
Functional Anatomy of Phospholipid Binding and Regulation of Phosphoinositide Homeostasis by Proteins of the Sec14 SuperfamilySchaaf, G.; Ortlund, E.; Tyeryar, K.; Mousley, Carl; Ile, K.; Garrett, T.; Ren, J.; Woolls, M.; Raetz, C.; Redinbo, M.; Bankaitis, V. (2008)Sec14, the major yeast phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein, regulates essential interfaces between lipid metabolism and membrane trafficking from the trans-Golgi network (TGN). How ...
Maffucci, T.; Falasca, Marco (2014)Phosphoinositides (PIs) are minor lipid components of cellular membranes that play critical roles in membrane dynamics, trafficking, and cellular signaling. Among the seven naturally occurring PIs, the monophosphate ...