| Abstract
| - Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentiallybinds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids betweenmembranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITPα and PITPβ isoforms(269−270 residues). The crystal structure of rat PITPβ complexed to dioleoylphosphatidylcholine wasdetermined to 2.18 Å resolution with molecular replacement using rat PITPα (77% sequence identify) asthe phasing model. A structure comparison of the α and β isoforms reveals minimal differences in proteinconformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solventstructure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using smallunilamellar vesicles with carefully controlled phospholipid composition, indicates that the β isoformshave minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles containpredominantly 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 PITPβ isoform clearly has themost diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutationsof two locations within the lipid binding cavity that possess differing biochemical properties werecharacterized: I84α/F83β and F225α/L224β. The 225/224 locus is more critical in determining substratespecificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specificactivity became PITPα (F225L) ≈ PITPβ and PITPβ (L224F) ≈ PITPα. The 225α/224β locus plays amodest role in the specificity of both isoforms toward CerPCho.
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