The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket
Résumé
Oxysterol binding protein (OSBP) homologues, ORPs, constitute a 12-member family in mammals. We employed an in vitro [ 3}H]25-hydroxycholesterol (25OH) binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [ 3}H]photo-25-hydroxycholesterol (photo25OH) and [ 3}H]photo-cholesterol (photoCH), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 were in vitro shown to bind 25OH. Glutathione-S-transferase (GST) fusions of the ORP1L (long) and S (short) variants bound 25OH with similar affinity (ORP1L, K d} 9.7 x 10 -8}M; ORP1S, K d} 8.4 x 10 -8}M), while the affinity of GST-ORP2 for 25OH was lower (K d} 3.9 x 10 -6}M). Molecular modeling suggested that ORP2 has a sterol binding pocket similar to that of S. cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of I249 by tryptophan or K150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand binding domains of the ORPs or the full-length proteins, photo25OH was bound to OSBP, ORP3, 4, 5, 6, 7, 8, 10 and 11. In addition, the ORP1L variant and full-length ORP3, 5, and 8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as oxysterol binding proteins and suggests that most of the mammalian ORPs are able to bind sterols.
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