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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,...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057712/ https://www.ncbi.nlm.nih.gov/pubmed/21248202 http://dx.doi.org/10.1091/mbc.E10-11-0903 |
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author | 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. |
author_facet | 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. |
author_sort | Schaaf, Gabriel |
collection | PubMed |
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. |
format | Text |
id | pubmed-3057712 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-30577122011-05-30 Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution 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. Mol Biol Cell Articles 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. The American Society for Cell Biology 2011-03-15 /pmc/articles/PMC3057712/ /pubmed/21248202 http://dx.doi.org/10.1091/mbc.E10-11-0903 Text en © 2011 Schaaf et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. |
spellingShingle | Articles 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. Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution |
title | Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution |
title_full | Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution |
title_fullStr | Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution |
title_full_unstemmed | Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution |
title_short | Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution |
title_sort | resurrection of a functional phosphatidylinositol transfer protein from a pseudo-sec14 scaffold by directed evolution |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057712/ https://www.ncbi.nlm.nih.gov/pubmed/21248202 http://dx.doi.org/10.1091/mbc.E10-11-0903 |
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