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,...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Journal Article |
| Published: |
2011
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| Online Access: | http://hdl.handle.net/20.500.11937/9005 |
| _version_ | 1848745825533952000 |
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| author | Schaaf, G. Dynowski, M. Mousley, Carl Shah, S. Yuan, P. Winklbauer, E. De Campos, M. Trettin, K. Quinones, M. Smirnova, T. Yanagisawa, L. Ortlund, E. Bankaitis, V. |
| author_facet | Schaaf, G. Dynowski, M. Mousley, Carl Shah, S. Yuan, P. Winklbauer, E. De Campos, M. Trettin, K. Quinones, M. Smirnova, T. Yanagisawa, L. Ortlund, E. Bankaitis, V. |
| author_sort | Schaaf, G. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| 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. © 2011 Schaaf et al. |
| first_indexed | 2025-11-14T06:23:31Z |
| format | Journal Article |
| id | curtin-20.500.11937-9005 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T06:23:31Z |
| publishDate | 2011 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-90052017-09-13T14:49:27Z Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution Schaaf, G. Dynowski, M. Mousley, Carl Shah, S. Yuan, P. Winklbauer, E. De Campos, M. Trettin, K. Quinones, M. Smirnova, T. Yanagisawa, L. Ortlund, E. Bankaitis, V. 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. 2011 Journal Article http://hdl.handle.net/20.500.11937/9005 10.1091/mbc.E10-11-0903 unknown |
| spellingShingle | Schaaf, G. Dynowski, M. Mousley, Carl Shah, S. Yuan, P. Winklbauer, E. De Campos, M. Trettin, K. Quinones, M. Smirnova, T. Yanagisawa, L. Ortlund, E. Bankaitis, V. 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 |
| url | http://hdl.handle.net/20.500.11937/9005 |