Structure of PITPβ in complex with phosphatidylcholine:: Comparison of structure and lipid transfer to other PITP isoforms

Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentially binds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids between membranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITP alpha and PITP beta isoforms (269-270 residues). The crystal structure of rat PITP beta complexed to dioleoylphosphatidylcholine was determined to 2.18 angstrom resolution with molecular replacement using rat PITP alpha (77% sequence identify) as the phasing model. A structure comparison of the alpha and beta isoforms reveals minimal differences in protein conformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solvent structure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using small unilamellar vesicles with carefully controlled phospholipid composition, indicates that the isoforms have minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles contain predominantly PtdCho. When PtdCho and PtdIns are present in equivalent concentrations in donor vesicles, PtdIns transfer occurs at approximately 3-fold the rate of PtdCho. The rat PITP beta isoform clearly has the most diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutations of two locations within the lipid binding cavity that possess differing biochemical properties were characterized: I84 alpha/F83 beta and F225 alpha/L224 beta. The 225/224 locus is more critical in determining substrate specificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specific activity became PITP alpha (F225L) approximate to PITP beta and PITP beta (L224F) approximate to PITP alpha. The 225 alpha/224 beta locus plays a modest role in the specificity of both isoforms toward CerPCho.