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 of high density lipoprotein (HDL) metabolism. We have here, based on sequence alignments of the plasma LPS-binding/lipid transfer protein family and the X-ray structure of the bactericidal/permeability increasing protein (BPI), modeled the structure of PLTP. The model predicts a two-domain architecture with conserved lipid-binding pockets consisting of apolar residues in each domain. By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets are shown to be essential for PLTP-mediated phospholipid transfer. A solid phase ligand binding assay was used to determine the HDL-binding ability of the mutants. The results suggest that the observed decreases in phospholipid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDL-binding, but the C-terminal lipid-binding pocket may be involved in the association of PLTP with HDL. Further, the essential structural role of a disulfide bridge between cysteine residues 146 and 185 is demonstrated. The structural model and the mutants characterized here provide powerful tools for the detailed analysis of the mechanisms of PLTP function.
Huuskonen
J
J
Department of Biochemistry, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland.
Wohlfahrt
G
G
Jauhiainen
M
M
Ehnholm
C
C
Teleman
O
O
Olkkonen
V M
VM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism; D053440:Lipoproteins, HDL3; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D004220:Disulfides; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids