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Author Notes:

Address correspondence to: Vytas A. Bankaitis: vytas@med.unc.edu or Gabriel Schaaf: gabriel.schaaf@zmbp.uni-tuebingen.de.

Contact information for MD simulation data, ed.bew@ikswonyd-keram; EPR data, ude.uscn@onrimsit; and structural determination, eric.ortland@emory.edu.


Research Funding:

This work was supported by grant GM44530 from the National Institutes of Health to V.A.B., and it supported the efforts of G.S., C.J.M., S.D.S., P. Y., K. T., and L.L.Y. G. S., M. D., E.M.W., and M.K.F.C. are supported by grants SCHA 1274/1-1 and Emmy Noether grant 1274/2-1 from the Deutsche Forschungsgemeinschaft, T. S. and M.C.Q. are supported by NSF-0843632, and E. O. is supported by Emory University Start-up Funds. We acknowledge the University of North Carolina Lineberger Comprehensive Cancer Center Genome Analysis and Nucleic Acids Core facilities.

We also thank bwGRID, a member of the German D-Grid initiative, founded by the Ministry for Education and Research and the Ministry for Science, Research, and Arts Baden-Württemberg, for providing computational resources (www.bw-grid.de).


  • Science & Technology
  • Life Sciences & Biomedicine
  • Cell Biology
  • SEC14P
  • SAC1

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

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Journal Title:

Molecular Biology of the Cell


Volume 22, Number 6


, Pages 892-905

Type of Work:

Article | Final Publisher PDF


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.

Copyright information:

© 2011 Schaaf et al.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).

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