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Article No. jmbi.1999.3355 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 295, 595±603 NMR Structure of the Sterol Carrier Protein-2: Implications for the Biological Role Francisco LoÂpez GarcõÂa{1, Thomas Szyperski{1, James H. Dyer2 Thomas Choinowski2, Udo Seedorf3, Helmut Hauser2 and Kurt WuÈthrich1* 1 Institut fuÈr Molekularbiologie und Biophysik, EidgenoÈssische Technische HochschuleHoÈnggerberg, CH-8093 ZuÈrich Switzerland 2 Institut fuÈr Biochemie EidgenoÈssische Technische Hochschule-Zentrum CH-8092 ZuÈrich Switzerland 3 Institut fuÈr Arterioskleroseforschung WestfaÈlische WilhelmsUniversitaÈt, MuÈnster, Germany *Corresponding author The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone 15N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for investigations of the mode of action of SCP2. The SCP2 fold is formed by a ®ve-stranded b-sheet and four a-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the ®rst and third helices, and the b-sheet, which are all located in the polypeptide segment 8-102, was identi®ed with the use of the spinlabeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding. # 2000 Academic Press Keywords: sterol carrier protein 2; NMR; protein structure; protein dynamics; nitroxide spin labels Introduction The sterol carrier protein-2 (SCP2), or ``nonspeci®c lipid transport protein'' (Bloj & Zilversmit, 1997) is a ubiquitous 123-residue protein that has long been assumed to function in intracellular traf®cking. It is found in a wide range of tissues, and since its primary structure is highly conserved in different species (Seedorf & Assmann, 1991; Yamamoto et al., 1991; Monccechi et al., 1991; Pfeifer et al., 1993), it is believed to play an important role in lipid metabolism (Weber et al., 1998; Stolowich et al., 1997). Other proteins that carry C-terminal SCP2 domains include the 58 kDa peroxisomal thiolase (also called sterol carrier protein-X, SCPX) (Seedorf et al., 1994b), the peroxisomal D-hydroxyacyl-CoA dehydrogenase (also known as 17b- hydroxysteroid dehydrogenase type 4) (Leenders et al., 1996) and the Caenorhabditis elegans behavioral protein (unc-24) (Barnes et al., 1996). In all these proteins, the C-terminal SCP2 domains are involved in lipid binding and targeting of the proteins to the proper intracellular location. Gene targeting in mice has shown that SCP2 de®ciency results in peroxisome proliferation and severe impairment of the peroxisomal pathway (Seedorf et al., 1998) involved in the oxidation of a and b-methylated acyl-coenzyme A (CoA) esters such as phytanoyl-CoA, pristanoyl-CoA and trihydroxycholestanoyl-CoA. Since SCP2 de®ciency is furthermore associated with chronic superstimulation of the peroxisomal proliferator-activated receptor a (PPARa) (Ellinghaus et al., 1999) and spontaneous hepatocarcinogenesis (U.S., unpublished results), SCP2 is assumed to {These two authors contributed equally to the paper. Present address: Dr Thomas Szyperski, Department of Chemistry, 816 Natural Sciences Complex, State University of New York at Buffalo, Buffalo, NY 14260, USA. Abbreviations used: SCP2, sterol carrier protein-2; SCPX, sterol carrier protein-X; hSCP2, human SCP2 with residues 1-123; rSCP2, rabbit SCP2; CoA, coenzyme A; PPARa, peroxisomal proliferator-activated receptor a; 16DA, 16-doxylstearic acid; PTS1, peroxisomal targeting signal 1; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; COSY, correlation spectroscopy; TOCSY, total correlation spectroscopy; ppm, parts per million; 3JNHa, vicinal spin-spin coupling constant between the backbone amide proton and the a-proton; 3JNb, vicinal spin-spin coupling constant between the backbone amide nitrogen and one of the b-protons; 3Jab, vicinal spin-spin coupling constant between the a-proton and one of the b-protons. 0022-2836/00/030595±9 $35.00/0 # 2000 Academic Press