The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket

Abstract

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH 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, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

“…The sterol-binding domain of ORP9 is homologous to OSBP and other ORPs yet was reported not to bind photoderivatives of 25-hydroxycholesterol and cholesterol (Suchanek et al, 2007). As well, it is unclear if ORP9L or OSBP can transfer sterols from donor to acceptor membranes.…”

“…Binding of OSBP and ORP9L to phosphatidylcholine (PC) liposomes containing increasing PI-4P was determined by sedimentation ( Figure 1D). By this method, ϳ50% of ORP9L was associated with liposomes containing 10 mol% PI-4P, compared with 80 -90% for OSBP.The sterol-binding domain of ORP9 is homologous to OSBP and other ORPs yet was reported not to bind photoderivatives of 25-hydroxycholesterol and cholesterol (Suchanek et al, 2007). As well, it is unclear if ORP9L or OSBP can transfer sterols from donor to acceptor membranes.…”

“…The ORP9 gene encodes a full-length (ORP9L) and truncated version (ORP9S) that initiates from an alternate promoter and is missing the N-terminal PH domain (Wyles and Ridgway, 2004). Both ORP9S and ORP9L contain a FFAT motif that binds VAP and an OSH domain with undefined sterolbinding specificity (Suchanek et al, 2007). ORP9L partitioned between the ER and Golgi apparatus in a VAPdependent manner, and enforced overexpression disrupted the organization of the ER and ER-Golgi intermediate compartment (Wyles and Ridgway, 2004).…”

OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids

“…We recently reported that ORP2 binds 25OHC with micromolar affinity (31). We now wanted to study the oxysterol binding properties of ORP2 more extensively.…”

Genetic Control of Survival and Weight Loss during Pneumonic Burkholderia pseudomallei (Bp) Infection

“…The gene product of Ss18l1 (Chr 2) is a transcriptional activator that is required for calcium-dependent dendritic growth and branching in cortical neurons. Psma7 (Chr 2) encodes a member of the peptidase T1A family that functions as a 20S core alpha proteasome subunit and participates in cytoplasmic protein degradation and recycling, while OsBpl2 encodes a member of the oxysterol-binding protein family of intracellular lipid receptors, known as oxysterol binding protein-like 2 [139]. Two of the high-priority candidate genes on chromosome 17, Park2 and Pacrg, share a bidirectional promoter and are co-regulated in various tissues.…”