title: Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor.
abstract: We present the crystal structures of the SEC14-like domain of supernatant protein factor (SPF) in complex with squalene and 2,3-oxidosqualene. The structures were resolved at 1.75Å (complex with squalene) and 1.6Å resolution (complex with 2,3-oxidosqualene), leading in both cases to clear images of the protein/substrate interactions. Ligand binding is facilitated by removal of the Golgi-dynamics (GOLD) C-terminal domain of SPF, which, as shown in previous structures of the apo-protein, blocked the opening of the binding pocket to the exterior. Both substrates bind into a large hydrophobic cavity, typical of such lipid-transporter family. Our structures report no specific recognition mode for the epoxide group. In fact, for both molecules, ligand affinity is dominated by hydrophobic interactions, and independent investigations by computational models or differential scanning micro-calorimetry reveal similar binding affinities for both ligands. Our findings elucidate the molecular bases of the role of SPF in sterol endo-synthesis, supporting the original hypothesis that SPF is a facilitator of substrate flow within the sterol synthetic pathway. Moreover, our results suggest that the GOLD domain acts as a regulator, as its conformational displacement must occur to favor ligand binding and release during the different synthetic steps.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Monika
authors.initials: M
authors.lastname: Christen
authors.affiliation: École Polytechnique Fédéral Lausanne, Station 19, CH-1015 Lausanne, Switzerland.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Marcaida
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Christos
authors.initials: C
authors.lastname: Lamprakis
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Walter
authors.initials: W
authors.lastname: Aeschimann
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Jathana
authors.initials: J
authors.lastname: Vaithilingam
authors.affiliation: Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 4, CH-8093 Zürich, Switzerland.
authors.forename: Petra
authors.initials: P
authors.lastname: Schneider
authors.affiliation: Laboratory of Biomolecular Research (LBR), Paul Scherrer Institut, Villigen-PSI CH-5232, Switzerland.
authors.forename: Manuel
authors.initials: M
authors.lastname: Hilbert
authors.affiliation: Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg 4, CH-8093 Zürich, Switzerland.
authors.forename: Gisbert
authors.initials: G
authors.lastname: Schneider
authors.affiliation: Department of Chemistry and Centre for Theoretical and Computational Chemistry (CTCC), University of Oslo, P.O. Box 1033 Blindern, N-0315 Oslo, Norway. Electronic address: michele.cascella@kjemi.uio.no.
authors.forename: Michele
authors.initials: M
authors.lastname: Cascella
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland. Electronic address: achim.stocker@dcb.unibe.ch.
authors.forename: Achim
authors.initials: A
authors.lastname: Stocker
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D013185:Squalene / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
chemical_list: C002821:2,3-oxidosqualene; D002352:Carrier Proteins; D008024:Ligands; D013185:Squalene; D002784:Cholesterol
keywords: CRAL-TRIO; Cholesterol biosynthesis; Free energy perturbation; Lipid binding; SEC-14 like