Ligand Specificity in the CRAL-TRIO Protein Family

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Abstract
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 α-tocopherol transfer protein (α-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 [3H]-RRR-α-tocopherol binding assay. α-TTP preferentially bound RRR-α-tocopherol over all other tocols assayed, exhibiting a Kd of 25 nM. Binding affinities of other tocols for αTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays. All other homologous proteins studied bound α-tocopherol but with pronouncedly weaker (> 10-fold) affinities than α-TTP. Sec14p demonstrated a Kd of 373 nM for α-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM). Human SPF had the highest affinity for phosphatidylinositol (216 nM) and γ-tocopherol (268 nM) and significantly weaker affinity for α-tocopherol (Kd 615 nM). SPF bound [3H]-squalene more weakly (879 nM) than the other ligands. Our data suggest that of all known CRAL-TRIO proteins, only αTTP is likely to serve as the physiological mediator of α-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.