Sterol carrier protein-2: structure reveals function

Abstract

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the ...

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

“…We found that there was a striking difference in the binding kinetics of these ligands, oleate having a 15-fold greater k a and a 2-fold greater k d , giving a dissociation equilibrium constant (K d ) 9-times lower than that of linoleate. Monoenoic oleate is less hydrophilic than dienoic linoleate and this could possibly explain its higher on-rate and lower K d value, given that fatty acid binding occurs in a relatively hydrophobic pocket of SCP-2 [8]. It is worth noting that the K d we determined for oleate (~220 nM) is within range of the values previously reported for fluorophore-labeled stearate, e.g.…”

“…1, FPLC-purified recombinant SCP-2 appeared as a single band on SDS-PAGE, its M r being ~13 kDa, as expected [7,8,11]. Expressed bSCP-2 isolated by SoftLink affinity chromatography also migrated as a single band (Fig.…”

“…Isolated naturally occurring or recombinant SCP-2 is known to interact with a wide variety of lipids, including fatty acids, fatty acyl-CoAs, phospholipids, and sterols [7,8]. Heretofore, these interactions have typically been studied using lipids conjugated with some type of relatively polar reporter group [7,16,17].…”

“…Among the three basic types of transfer protein identified in mammalian cells [3], 13.2 kDa sterol carrier protein-2 (SCP-2) has been studied most extensively since its isolation about 25 years ago [2]. SCP-2 is encoded by the SCP-x/pro-SCP-2 composite gene, expression of which gives 58 kDa SCP-x (~45 kDa representing peroxisomal 3-ketoacyl-CoA thiolase) and 15.2 kDa pro-SCP-2 [7,8]. Mature SCP-2 can be generated by post-translational C-terminal proteolysis of either pro-SCP-2 or SCP-x [7,8].…”

“…SCP-2 is encoded by the SCP-x/pro-SCP-2 composite gene, expression of which gives 58 kDa SCP-x (~45 kDa representing peroxisomal 3-ketoacyl-CoA thiolase) and 15.2 kDa pro-SCP-2 [7,8]. Mature SCP-2 can be generated by post-translational C-terminal proteolysis of either pro-SCP-2 or SCP-x [7,8]. Also known as non-specific lipid transfer protein because it recognizes phospholipids, fatty acids, and fatty acyl CoAs in addition to cholesterol, SCP-2 is found in several subcellular compartments, most prominently in peroxisomes, but also in mitochondria, lysosomes, and cytosol [7,9].…”