Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor

Michele Cascella, Christos Lamprakis, Maria Josefina Marcaida Lopez
Academic Press Inc Elsevier Science, 2015
Article

Résumé

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 1.75 angstrom (complex with squalene) and 1.6 angstrom 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-calorimetly 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. (C) 2015 Elsevier Inc. All rights reserved.

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Michele Cascella, Christos Lamprakis, Maria Josefina Marcaida Lopez
Academic Press Inc Elsevier Science, 2015
Article

Résumé

Official source

https://infoscience.epfl.ch/record/212559?ln=fr

À propos de ce résultat

Concepts associés (13)

Concepts associés

Chargement

Publications associées (2)

Publications associées

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From weakly coordinating to non-coordinating anions? A simple preparation of the silver salt of the least coordinating anion and its application to determine the ground state structure of the Ag(η2-P4)2+ cation

Ines Raabe, Nils Trapp

The unexpected but facile prepn. of the Ag salt of the least coordinating [(RO)3Al-F-Al(OR)3]- anion (R = C(CF3)3) by reaction of Ag[Al(OR)4] with one equiv. of PCl3 is described. The mechanism of the formation of Ag[(RO)3Al-F-Al(OR)3] is explained based on the available exptl. data as well as on quantum chem. calcns. with the inclusion of entropy and COSMO solvation enthalpies. The crystal structures of (RO)3Al(OC4H8), Cs[(RO)2(Me)AlFAl(Me)(OR)2], Ag(CH2Cl2)3[(RO)3AlFAl(OR)3] and Ag(h2-P4)2[(RO)3AlFAl(OR)3]- are described. From the collected data the [(RO)3AlFAl(OR)3]- anion is the least coordinating anion currently known. With respect to the F- ion affinity of two parent Lewis acids Al(OR)3 of 685 kJ mol-1, the ligand affinity (441 kJ mol-1), the proton and Cu decompn. reactions (-983 and -297 kJ mol-1) as well as HOMO level and HOMO-LUMO gap and in comparison with [Sb4F21]-, [Sb(OTeF5)6]-, [Al(OR)4]- as well as [B(RF)4]- (RF = CF3 or C6F5) the [(RO)3Al-F-Al(OR)3]- anion is among the best weakly coordinating anions (WCAs) according to each value. In contrast to most of the other cited anions, the [(RO)3AlFAl(OR)3] anion is available by a simple prepn. in conventional inorg. labs. The least coordinating character of this anion was employed to clarify the question of the ground state geometry of the Ag(h2-P4)2+ cation (D2h, D2 or D2d). In agreement with computational data and NMR spectra it could be shown that the rotation along the Ag-(P-P-centroid) vector has no barrier and that the structure adopted in the solid state depends on packing effects which lead to an almost D2h sym. Ag(h2-P4)2+ cation (0 to 10.6 Deg torsion) for the more sym. [Al(OR)4]- anion, but to a D2 sym. Ag(h2-P4)2+ cation with a 44 Deg twist angle of the two AgP2 planes for the less sym. [(RO)3AlFAl(OR)3]- anion. This implies that Ag back bonding, suggested by quantum chem. population analyses to be of importance, is only weak. [on SciFinder (R)]

2004

Chargement

Molecular interactions of odorants with their olfactory receptors (ORs) are of central importance for the mammalian olfactory system to detect and discriminate a large variety of odors with a limited set of receptors. How a particular OR binds and distinguishes different odorant molecules remains unknown on a structural basis as there is no three- dimensional OR structure available. Progress in the area of ligand assignment to specific receptors with the development of functional assays allowing efficient screening of numerous odorant compound libraries is essential for the understanding of the selectivity and sensitivity of odor detection. To improve our knowledge of an OR binding site environment, the first part of this thesis focused on the characterization of the molecular interactions of the mouse eugenol receptor (mOR-EG) which has been shown to be activated by a wide variety of odorants. Computer modeling based on the resolved 3D structure of the β2-adrenergic receptor and mutational studies on mOR-EG were used to determine the key amino acid residues responsible for specific OR-ligand recognition and to determine the localization and chemical properties of the putative ligand binding site. Activation of mOR-EG point mutants was monitored using a cAMP-induced reporter gene assay in Hana3A cells. These investigations were used to improve ligand docking in the binding site and further define a more precise model of the mOR-EG ligand-binding cavity. With the development of genome sequencing, the comparison of ortholog/paralog pairs is becoming increasingly important to study the molecular basis of OR specificity. Emphasis has thus been placed on the characterization of the mOR-EG’s mouse paralog mOR-EV and on its putative human ortholog hOR5D18. These two ORs have been functionally expressed in Hana3A cells and the visualization of both their overall- and cell-surface expression have been performed using different OR fusion proteins. New specific agonists for the mOR-EV have been identified (jasmone, rose oxyde and nootkatone) and first attempts to de-orphanize hOR5D18 have been carried out, by random screening a large panel of more than 250 odorant compounds coupling the OR activation to cAMP-induced reporter gene expression. Based on our knowledge of the mOR-EG ligand binding site, three important positions for ligand recognition that diverge between mOR-EG and mOR-EV were mutated in both receptors to increase their sequence identity. Comparison of their respective ligand repertoires has provided novel insight into the molecular determinants for odor detection and discrimination. Given the importance to assign ligands to their cognate receptors, we established a novel functional assay based on electrical impedance measurements. The detection of ligand-induced cell morphological changes during and after receptor stimulation has been used to quantify the activation of different GPCRs including mOR-EG with their respective ligands. This label-free technique is applicable for many different cell types as well as for endogenously expressed receptors and is suitable for screening large libraries of ORs and compounds. Odorants are generally small hydrophobic cell-membrane-permeable organic molecules with highly variable chemical structures and properties. Due to their large chemical diversity, it is likely that some of them might trigger other cellular processes apart from their "conventional" role in olfaction. The dual role of some odorants acting on the mOR-EG and/or the nuclear estrogen receptor α (ERα) has therefore been explored. Estrogenic effect of these odorants has been measured on ER endogenously expressing MCF7 cells, which represent a sensitive and well-established system for the detection of estrogenic activities, using a Luciferase reporter gene assay and different cell proliferation measuring methods. Finally, computational modeling and ligand docking have provided informations on the molecular recognition principles of odorants on two functionally non-related receptors and will therefore help for the evaluation of the potential risk of chemical compounds used in perfumery.

EPFL2012

Chargement

From weakly coordinating to non-coordinating anions? A simple preparation of the silver salt of the least coordinating anion and its application to determine the ground state structure of the Ag(η2-P4)2+ cation

Ines Raabe, Nils Trapp

2004

EPFL2012