Ligand specificity in the CRAL-TRIO protein family.
Intracellular trafficking of hydrophobic ligands is often mediated by specific binding proteins. The CRAL-TRIO motif is common to several lipid binding proteins including the cellular retinaldehyde binding protein (CRALBP), the alpha-tocopherol transfer protein (alpha-TTP), yeast phosphatidylinositol transfer protein (Sec14p), and supernatant protein factor (SPF). To examine the ligand specificity of these proteins, we measured their affinity toward a variety of hydrophobic ligands using a competitive [(3)H]-RRR-alpha-tocopherol binding assay. Alpha-TTP preferentially bound RRR-alpha-tocopherol over all other tocols assayed, exhibiting a K(d) of 25 nM. Binding affinities of other tocols for alphaTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays. All other homologous proteins studied bound alpha-tocopherol but with pronouncedly weaker (> 10-fold) affinities than alpha-TTP. Sec14p demonstrated a K(d) of 373 nM for alpha-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM). Human SPF had the highest affinity for phosphatidylinositol (216 nM) and gamma-tocopherol (268 nM) and significantly weaker affinity for alpha-tocopherol (K(d) 615 nM). SPF bound [(3)H]-squalene more weakly (879 nM) than the other ligands. Our data suggest that of all known CRAL-TRIO proteins, only alphaTTP is likely to serve as the physiological mediator of alpha-tocopherol's biological activity. Further, ligand promiscuity observed within this family suggests that caution should be exercised when suggesting protein function(s) from measurements utilizing a single ligand.
Panagabko
Candace
C
Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, Ontario, Canada, L2S 3A1.
Morley
Samantha
S
Hernandez
Marta
M
Cassolato
Patrick
P
Gordon
Heather
H
Parsons
Rachel
R
Manor
Danny
D
Atkinson
Jeffrey
J
D000465:Algorithms; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008055:Lipids / Q000737:chemistry; D008074:Lipoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010716:Phosphatidylinositols / Q000737:chemistry*; D049294:Phospholipid Transfer Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D014316:Tritium / Q000494:pharmacology; D024502:alpha-Tocopherol / Q000737:chemistry; D024504:gamma-Tocopherol / Q000737:chemistry
D002352:Carrier Proteins; D008024:Ligands; D008055:Lipids; D008074:Lipoproteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins; C432422:SEC14L2 protein, human; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C017763:alpha-tocopherol transfer protein; D014316:Tritium; D024504:gamma-Tocopherol; D024502:alpha-Tocopherol