3Q8G

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

Summary for 3Q8G

• Entry DOI: 10.2210/pdb3q8g/pdb
• Related: 3B74 3B7N 3B7Q 3B7Z
• Descriptor: CRAL-TRIO domain-containing protein YKL091C, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, GLYCEROL, ... (4 entities in total)
• Functional Keywords: cral-trio, string motif, signaling protein, directed evolution, pipt, sec14, phospholipid transporter, lipid, phosphatidylinositol, phosphatidylcholine
• Biological source: Saccharomyces cerevisiae (yeast)
• Total number of polymer chains: 1
• Total formula weight: 38191.85

Authors

Ortlund, E.A.,Schaaf, G.,Bankaitis, V.A. (deposition date: 2011-01-06, release date: 2011-02-23, Last modification date: 2024-05-22)

Primary citation

Schaaf, G.,Dynowski, M.,Mousley, C.J.,Shah, S.D.,Yuan, P.,Winklbauer, E.M.,de Campos, M.K.,Trettin, K.,Quinones, M.C.,Smirnova, T.I.,Yanagisawa, L.L.,Ortlund, E.A.,Bankaitis, V.A.
Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution.
Mol.Biol.Cell, 22:892-905, 2011

PubMed Abstract: 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.

PubMed: 21248202
DOI: 10.1091/mbc.E10-11-0903

Experimental method

X-RAY DIFFRACTION (1.8 Å)