title: The chemistry of phospholipid binding by the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p as determined by EPR spectroscopy.
abstract: The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA. Tatyana_Smirnova@ncsu.edu
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Smirnova
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Chadwick
authors.forename: Ryan
authors.initials: R
authors.lastname: MacArthur
authors.forename: Oleg
authors.initials: O
authors.lastname: Poluektov
authors.forename: Likai
authors.initials: L
authors.lastname: Song
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Ryan
authors.forename: Gabriel
authors.initials: G
authors.lastname: Schaaf
authors.forename: Vytas A
authors.initials: VA
authors.lastname: Bankaitis
mesh_terms: D001665:Binding Sites; D005786:Gene Expression Regulation; D008958:Models, Molecular; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; C483438:SEC14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins