Crystal Structure of the Liganded SCP-2-like Domain of Human Peroxisomal Multifunctional Enzyme Type 2 at 1.75 A Ê Resolution Antti M. Haapalainen 1 , Daan M. F. van Aalten 2 , Gitte Merila È inen 1 Jorma E. Jalonen 3 , Pa È ivi Pirila È 1 , Rik K. Wierenga 1 , J. Kalervo Hiltunen 1 and Tuomo Glumoff 1 * 1 Biocenter Oulu and Department of Biochemistry University of Oulu, P.O. Box 3000, FIN-90014, University of Oulu, Finland 2 Wellcome Trust Biocentre, School of Life Sciences University of Dundee, Dundee DD1 5EH, UK 3 Department of Chemistry University of Oulu, P.O. Box 3000, FIN-90014, University of Oulu, Finland b-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl- CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral speci®city. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid resi- dues 618-736 of human MFE-2 (dhSCP-2L) was solved at 1.75 A Ê resol- ution in complex with Triton X-100, an analog of a lipid molecule. This is the ®rst reported structure of an MFE-2 domain. The dhSCP-2L has an a/b-fold consisting of ®ve b-strands and ®ve a-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of dhSCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solvent- exposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism. # 2001 Academic Press Keywords: multifunctional enzyme; b-oxidation; peroxisome; Triton X-100; sterol *Corresponding author Introduction All the characterized b-oxidation pathways are found to have a multifunctional enzyme (MFE). A common feature of these MFEs is that they catalyze the second and the third reaction of the pathway. The properties of the MFEs, like subunit compo- sition and associated enzymatic activities, vary depending on species and whether they originate from mitochondria, peroxisomes (eukaryotes) or from the cytoplasm (bacteria). 1 Multifunctional enzyme type 2 (MFE-2, EC 1.1.1.62) in mammals, also known as D-bifunctional protein }2 and 17b- hydroxysteroid dehydrogenase type 4 (17b-HSD 4), 3 metabolizes trans-2-enoyl-CoA esters to 3-keto compounds via (3R)-hydroxyacyl-CoA intermedi- ates in the peroxisomal b-oxidation. MFE-2 has been cloned and characterized from a wide range of organisms. 4±8 Analysis of accumulating metab- olites in patients with MFE-2 de®ciency and ``knock-out'' mice, together with enzymatic proper- ties observed in vitro, suggest that the physiological E-mail address of the corresponding author: Tuomo.Glumoff@oulu.® Abbreviations used: MFE, multifunctional enzyme; MFE-2, multifunctional enzyme type 2; dhSCP-2L, MFE-2 truncated for (3R)-hydroxyacyl-CoA dehydrogenase and trans-2-enoyl-CoA hydratase 2 domains; SCP-2L, sterol carrier protein type 2-like domain; SCP-2, sterol carrier protein type 2; SCPX, sterol carrier protein x; C6, fatty acid molecule with six carbon atoms; CoA, coenzyme A; PTS1, peroxisomal targeting signal type 1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. doi:10.1006/jmbi.2001.5084 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 313, 1127±1138 0022-2836/01/051127±12 $35.00/0 # 2001 Academic Press