Crystallization and preliminary X-ray diffraction analysis of phospholipid-bound Sfh1p, a member of the Saccharomyces cerevisiae Sec14p-like phosphatidylinositol transfer protein family.
Sec14p is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the budding yeast Saccharomyces cerevisiae and is the founding member of a large eukaryotic protein superfamily. This protein catalyzes the exchange of either PtdIns or PtdCho between membrane bilayers in vitro and this exchange reaction requires no external input of energy or of other protein cofactors. Despite the previous elucidation of the crystal structure of a detergent-bound form of Sec14p, the conformational changes that accompany the phospholipid-exchange reaction remain undefined. Moreover, a structural appreciation of how Sec14p or its homologs bind their various phospholipid substrates remains elusive. Here, the purification and crystallization of yeast Sfh1p, the protein most closely related to Sec14p, are reported. A combination of electrospray ionization mass-spectrometry and collision-induced decomposition mass-spectrometry methods indicate that recombinant Sfh1p loads predominantly with phosphatidylethanolamine. Unlike phospholipid-bound forms of Sec14p, this form of Sfh1p crystallizes readily in the absence of detergent. Sfh1p crystals diffract to 2.5 A and belong to the orthorhombic primitive space group P2(1)2(1)2(1), with unit-cell parameters a = 49.40, b = 71.55, c = 98.21 A, alpha = beta = gamma = 90 degrees. One Sfh1p molecule is present in the asymmetric unit (V(M) = 2.5 A(3) Da(-1); V(s) = 50%). Crystallization of a phospholipid-bound Sec14p-like protein is a critical first step in obtaining the first high-resolution picture of how proteins of the Sec14p superfamily bind their phospholipid ligands. This information will significantly extend our current understanding of how Sec14p-like proteins catalyze phospholipid exchange.
Schaaf
Gabriel
G
Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090, USA.
Betts
Laurie
L
Garrett
Teresa A
TA
Raetz
Christian R H
CR
Bankaitis
Vytas A
VA
D001483:Base Sequence; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D013058:Mass Spectrometry; D010716:Phosphatidylinositols / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; C483438:SEC14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C106163:Sfh1 protein, S cerevisiae