Structure and Ligands of ORPs

The ORPs minimally comprise an OSBP-related ligand-binding domain (ORD), but in mammals a majority of them carry an amino-terminal extension containing a pleckstrin homology (PH) domain that is in several cases known to bind membrane phosphoinositides (Johansson et al. 2005 ; Lehto et al. 2005 ; Levine and Munro 1998, 2002) . The proteins consisting of an ORD only are here designated “short ORPs”, while those carrying a PH domain are called “long ORPs”.

Identification of ligands for the ORDs of the ORPs is crucial for elucidation of the functions of these proteins. The first ORP high-resolution structure, that of a short yeast S. cerevisiae ORP, Osh4p/Kes1p, revealed that Osh4p is a sterol-binding protein (Im et al. 2005) . It was crystallized in complex with five different sterols, and has a sterol binding pocket formed by 19 b -strands in an antiparallel arrangement. The sheet bends to an almost complete roll that is, in the presence of bound ligand, closed by a lid containing an amphipathic a -helix connected by a flexible linker.

Sterols bind within the pocket oriented with the 3 b -hydroxyl group at the bottom of the hydrophobic binding tunnel. The sterol side-chain interacts with the lid, stabilizing its closed conformation. Importantly, many of the interactions of the bound sterol are mediated via water molecules within the pocket, giving the ligand interaction substantial flexibility. This provides a plausible explanation to the ability of the pocket to accommodate structurally different sterols and possibly also other types of lipid ligands. The structure of Osh4p suggested that this protein and its homologues might act as sterol transporters or mediators of sterol signals (Im et al. 2005) .

Of the mammalian ORPs, OSBP, ORP4 (also designated OSBP2), ORP1, and ORP2 have been shown to bind oxysterols (Moreira et al. 2001 ; Suchanek et al.

2007 ; Taylor et al. 1984 ; Wang et al. 2002 ; Yan et al. 2007a) . Moreover, ORP8 was reported to show affinity for 25-OHC (Yan et al. 2008) . Based on evidence obtained through the use of UV cross-linkable sterol derivatives in live cells, Suchanek et al.

(2007) suggested that a majority of the other human ORPs may bind sterols.

However, this data must be interpreted with caution since it is extremely difficult to verify the specificity of the live cell cross-linking signals. The structure of Osh4p

100 aa

Fig. 2.5 The human and S. cerevisiase OSBP-related protein ( ORP ) families. The major structural elements are identified. In humans, splice variation leading to changes in mRNA and protein structure is found for several ORPs (see NCBI database; Collier et al. 2003) . Therefore, the amino acid ( aa ) scale given is not exact but indicative; and the proteins depicted do not represent indi-vidual splice variants but rather groups formed by several variants

(Im et al. 2005) was used as template for molecular homology modeling of mam-malian ORP2 (Suchanek et al. 2007) and OSBP (Wang et al. 2008a) . In the former study, analysis of site-specific mutants designed based on the model suggested that ORP2 has a sterol-binding pocket similar to that of Osh4p. Interestingly, Wang et al. (2008a) showed evidence that the intact ORD of OSBP is not required for binding of 25-OHC or cholesterol, which is also known to be a ligand of OSBP (Wang et al. 2005c) . Specific and high-affinity sterol binding was also detected with a 51-amino-acid (aa) protein fragment that corresponds to the amino-terminal end of the ORD and comprises part of the modeled lid region (Fig. 2.6 ). One inter-pretation of the data is that the 51-aa segment forms the actual sterol binding site, and the b -barrel structure merely protects the bound sterol. The authors also found that a glycine/alanine-rich region at the amino-terminal end of OSBP, together with the PH domain, controls the binding of cholesterol but not of 25-OHC, suggesting interactions of the amino-terminal PH domain region and the ORD, most likely regulated by conformational changes triggered by ligand interactions. At the carboxy-terminal end of the ORD of OSBP and several other ORPs, there is a predicted

ORD (sterol binding)

1 77 88 182 732 761 809

PI(4)P (Golgi)

VAPs (ER)

HePTP Intramolecular

regulation

Pleckstrin homology (PH) domain Gly-Ala-rich region

Dimerization domain

ER targeting motif (FFAT) Sterol binding core sequence Coiled-coil domain

306 325 408 459 360

261 287 364

Leucine repeat motif Dimerization, vimentin regulation

Fig. 2.6 Structural elements important for OSBP function. The domains and sequence motifs are identified at the bottom. The pleckstrin homology (PH) domain specifies Golgi targeting mediated by PI(4)P and the ER targeting motif (FFAT) interacts with the VAP proteins, functions relevant for the control of sphingomyelin synthesis. OSBP forms homodimers and heterodimers with ORP4L, the latter process being connected with regulation of vimentin intermediate filament organization by ORP4L. A coiled-coil domain near the carboxy-terminus of OSBP interacts with HePTP, a tyrosine phosphatase that regulates the activity of extracellular signal regulated kinases, ERK. The Gly/Ala-rich region at the amino-terminus works with the PH domain to control cho-lesterol binding by the ORD, while occupancy of the ORD by oxysterol ligands regulates the Golgi-targeting function of the PH domain

coiled-coil forming region. Wang et al. (2008a) demonstrated that this region is required for the interaction of OSBP with a phosphatase acting on the ERK. The coiled-coil motif may play an important role in the protein–protein interactions of other ORPs as well (see Sect. 2.3.7 ).

In addition to sterol ligands, the ORDs of ORP1, ORP2, ORP9, and ORP10 have been suggested to show affinity for phosphoinositides (PIPs; Fairn and McMaster 2005a, b ; Hynynen et al. 2005) , but it is unclear whether these interactions involve a pocket such as that found in yeast Osh4p. Most likely, they represent binding of positively charged amino acid clusters on the protein surface to negatively charged membrane phosphoinositides (see Im et al. 2005) . The data by Raychaudhuri et al.

(2006) demonstrates that PIPs facilitate the transfer of sterols between membranes by Osh4p, suggesting that the PIP interaction plays an important regulatory role in the sterol sensor/transporter function of the short ORPs.