Holo-sterol carrier protein-2. (13)C NMR investigation of cholesterol and fatty acid binding sites.
Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Furthermore, the interrelationship(s) between this SCP-2 ligand binding site and the recently reported SCP-2 long chain fatty acid (LCFA) and long chain fatty acyl-CoA (LCFA-CoA) binding site(s) remains to be established. In the present work, two SCP-2 ligand binding sites were identified. First, both [4-(13)C]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-2. This ligand binding site had high affinity for NBD-cholesterol, K(d) = 4.15 +/- 0.71 nM. (13)C NMR-labeled ligand competition studies demonstrated that the SCP-2 high affinity cholesterol binding site also bound LCFA or LCFA-CoA. However, only the LCFA-CoA was able to effectively displace the SCP-2-bound [4-(13)C]cholesterol. Thus, the ligand affinities at this SCP-2 binding site were in the relative order cholesterol = LCFA-CoA > LCFA. Second, (13)C NMR studies demonstrated the presence of another ligand binding site on SCP-2 that bound either LCFA or LCFA-CoA but not cholesterol. Photon correlation spectroscopy was consistent with SCP-2 being monomeric in both liganded and unliganded states. In summary, both (13)C NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity binding site that bound cholesterol, LCFA, or LCFA-CoA. Furthermore, results with (13)C NMR supported the presence of a second SCP-2 ligand binding site that bound either LCFA or LCFA-CoA but not cholesterol. These data contribute to our understanding of a role for SCP-2 in both cellular cholesterol and LCFA metabolism.
Stolowich
N
N
Department of Chemistry, Texas A & M University, College Station, Texas 77843-4466, USA.
Frolov
A
A
Petrescu
A D
AD
Scott
A I
AI
Billheimer
J T
JT
Schroeder
F
F
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D001665:Binding Sites; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism* / Q000493:pharmacokinetics; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D019301:Oleic Acid / Q000378:metabolism; D010940:Plant Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D002247:Carbon Isotopes; D002352:Carrier Proteins; D005230:Fatty Acids, Nonesterified; D008024:Ligands; D010940:Plant Proteins; D011994:Recombinant Proteins; D012997:Solvents; C072087:sterol carrier proteins; D019301:Oleic Acid; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan