Structural and functional determinants of human plasma phospholipid transfer protein activity as revealed by site-directed mutagenesis of charged amino acids.
Human plasma phospholipid transfer protein (PLTP) exchanges phospholipids between lipoproteins and remodels high-density lipoproteins (HDLs). We determined phospholipid transfer activity and HDL binding ability in wild-type PLTP and in 16 PLTP variants created by replacing 12 charged amino acids by site-directed mutagenesis. The data were analyzed in relation to the structure of a member of the same gene family, bactericidal/permeability-increasing protein, which is a boomerang-shaped molecule containing two symmetrical, hydrophobic pockets that bind phospholipid molecules. When expressed in COS-7 cells, wild-type and all mutant PLTPs accumulated intracellularly to nearly the same extent. Relative to wild-type PLTP, substitution(s) for amino acids with a lateral position totally exposed to the solvent produced reductions in transfer activity proportional to the reductions in the level of HDL binding. Variants containing substitutions for charged amino acids on the concave surface of PLTP did not affect binding to HDL or specific transfer activity. A mutation in the C-terminal pocket (E270R) led to a decrease in both the specific transfer activity and the level of binding to HDLs, whereas mutations in the N-terminal pocket (R25E and D231R) resulted in a large decrease in specific transfer activity without affecting HDL binding. The data support a model of transfer in which N- and C-terminal pockets have different roles in HDL binding and transfer activity. The N-terminal pocket may be critical to PLTP transfer activity but may have no involvement in binding to lipoproteins, whereas amino acid substitutions in the C-terminal pocket might reduce PLTP activity by decreasing PLTP's affinity for HDLs.
Ponsin
Gabriel
G
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Qu
Shi-Jing
SJ
Fan
Hui-Zhen
HZ
Pownall
Henry J
HJ
D024342:Amino Acids, Acidic / Q000378:metabolism*; D024361:Amino Acids, Basic / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D049294:Phospholipid Transfer Proteins; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D002352:Carrier Proteins; D008075:Lipoproteins, HDL; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins