Holo-sterol carrier protein-2 - C-13 NMR investigation of cholesterol and fatty acid binding sites

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vit ro, almost nothing is known regarding the identity of the putative choleste rol 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 b e established. In the present work, two SCP-2 ligand binding sites were ide ntified, First, both [4-C-13]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1,3-d iazol-4-yl)amino)-23,24-bisnor-5-cholen-3 beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-S. Th is ligand binding site had high affinity for NBD-cholesterol, K-d = 4.15 +/ - 0.71 nm. C-13 NMR-labeled ligand competition studies demonstrated that th e 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-boun d [4-C-13]Cholesterol, Thus, the ligand affinities at this SCP-2 binding si te were in the relative order cholesterol = LCFA-CoA > LCFA. Second, C-13 N MR studies demonstrated the presence of another ligand binding site on SCP- 2 that bound either LCFA or LCFA-CoA but not cholesterol, Photon correlatio n spectroscopy was consistent with SCP-2 being monomeric in both liganded a nd unliganded states. In summary, both C-13 NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity bind ing site that bound cholesterol, LCFA, or LCFA-CoA, Furthermore, results wi th C-13 NMR supported the presence of a second SCP-2 ligand binding site th at 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.