Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis

The plasma phospholipid transfer protein (PLTP) is an important regulator o f high density lipoprotein (HDL) metabolism. We have here, based on sequenc e alignments of the plasma LPS-binding/lipid transfer protein family and th e X-ray structure of the bactericidal/permeability increasing protein (BPI) , modeled the structure of PLTP, The model predicts a two-domain architectu re with conserved lipid-binding pockets consisting of apolar residues in ea ch domain, By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets ar e shown to be essential for PLTP-mediated phospholipid transfer. A solid ph ase ligand binding assay was used to determine the HDL-binding ability of t he mutants,j/r The results suggest that the observed decreases in phospholi pid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDL- binding, but the C-terminal lipid-binding pocket may be in volved in the association of PLTP with HDL, Further, the essential structur al role of a disulfide bridge between cysteine residues 146 and 185 is demo nstrated. The structural model and the mutants characterized here provide p owerful tools for the detailed analysis of the mechanisms of PLTP function, , Structure and phospholipid transfer activity of human PLTP: analysis by m olecular modeling and site-directed mutagenesis.