Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 Å resolution 1 1Edited by R. Huber

Abstract

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-enoylCoA 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 residues 618-736 of human MFE-2 (dÁhÁSCP-2L) was solved at 1.75 A Ê resolution in complex with Triton X-100, an analog of a lipid molecule. This is the ®rst reported structure of an MFE-2 domain. The dÁhÁSCP-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 dÁhÁSCP-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 solventexposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…Each of the four variants, regardless of the presence or absence of either presequence or PTS1, were found by far-UV CD to be natively folded, as estimated by far-UV CD and confirming previous structural data [3,13,26,[29][30][31]. Previous spectroscopic studies [32,33] have shown that the presequence of SCP2 does not display regular secondary structure-a conclusion confirmed by the CD data presented here.…”

“…[26,30,31] in which the C-terminal segment of SCP2 is found to be in a coiled conformation. Crystal structures of liganded SCP2 [13,29] show the C-terminal segment adopting an a-helical structure ''capping'' the ligand binding pocket. This coil-helix transition upon ligand binding may to some degree explain the ''tightening'' of secondary structure we observe when SCP2 is loaded with stearoyl CoA.…”

Conformational Plasticity of the Lipid Transfer Protein SCP2

“…The lipid binding site utilized by all four ligands tested overlaps well with the hydrophobic cavity ( Figure 1A) and is consistent with the location of the phenyl moiety of Triton or C11-C16 of palmitic acid bound to SCP2 ( Author Manuscript (10,11). This central binding cavity likely represents a high-affinity target site that has general binding affinity for hydrophobic ligands (28).…”

“…Our NMR relaxation data ( Figure 4E,F, Supporting Information Figure 7) provide direct evidence for the presence of conformational flexibility (at picosecond to nanosecond time scales) in the SCP2 peptide backbone, which is also hinted by elevated X-ray B-factors in the C-terminal part of the SCP2 structures (10,13). Moreover, the CPMG/HSQC experiments ( Figure 5) indicate the presence of backbone dynamics also at slow (microsecond to millisecond) time scales.…”

“…Author manuscript; available in PMC 2016 September 08. (10,11). This central binding cavity likely represents a high-affinity target site that has general binding affinity for hydrophobic ligands (28).…”