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,...

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Veröffentlicht in:Molecular biology of the cell 2011-03, Vol.22 (6), p.892-905
Hauptverfasser: Schaaf, Gabriel, Dynowski, Marek, Mousley, Carl J, Shah, Sweety D, Yuan, Peihua, Winklbauer, Eva M, de Campos, Marília K F, Trettin, Kyle, Quinones, Mary-Chely, Smirnova, Tatyana I, Yanagisawa, Lora L, Ortlund, Eric A, Bankaitis, Vytas A
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container_end_page 905
container_issue 6
container_start_page 892
container_title Molecular biology of the cell
container_volume 22
creator Schaaf, Gabriel
Dynowski, Marek
Mousley, Carl J
Shah, Sweety D
Yuan, Peihua
Winklbauer, Eva M
de Campos, Marília K F
Trettin, Kyle
Quinones, Mary-Chely
Smirnova, Tatyana I
Yanagisawa, Lora L
Ortlund, Eric A
Bankaitis, Vytas A
description 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.
doi_str_mv 10.1091/mbc.e10-11-0903
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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.</abstract><cop>United States</cop><pub>The American Society for Cell Biology</pub><pmid>21248202</pmid><doi>10.1091/mbc.e10-11-0903</doi><tpages>14</tpages><oa>free_for_read</oa></addata></record>
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subjects Amino Acid Sequence
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - genetics
Cell Cycle Proteins - metabolism
Chromosomal Proteins, Non-Histone - chemistry
Chromosomal Proteins, Non-Histone - genetics
Chromosomal Proteins, Non-Histone - metabolism
Directed Molecular Evolution
Endosomes - metabolism
Golgi Apparatus - metabolism
Models, Molecular
Molecular Dynamics Simulation
Molecular Sequence Data
Phenotype
Phosphatidylcholines - chemistry
Phosphatidylcholines - metabolism
Phosphatidylinositols - chemistry
Phosphatidylinositols - metabolism
Phospholipid Transfer Proteins - chemistry
Phospholipid Transfer Proteins - genetics
Phospholipid Transfer Proteins - metabolism
Protein Conformation
Saccharomyces cerevisiae - cytology
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Sequence Alignment
Signal Transduction
trans-Golgi Network - metabolism
title Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution
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