Structure of human ORP3 ORD reveals conservation of a key function and ligand specificity in OSBP-related proteins

Abstract

Human ORP3 belongs to the oxysterol-binding protein (OSBP) family of lipid transfer proteins and is involved in lipid trafficking and cell signaling. ORP3 localizes to the ER-PM interfaces and is implicated in lipid transport and focal adhesion dynamics. Here, we report the 2.6–2.7 Å structures of the ORD (OSBP-related domain) of human ORP3 in apo-form and in complex with phosphatidylinositol 4-phosphate. The ORP3 ORD displays a helix grip β-barrel fold with a deep hydrophobic pocket which is conserved in the OSBP gene family. ORP3 binds PI(4)P by the residues around tunnel entrance and in the hydrophobic pocket, whereas it lacks sterol binding due to the narrow hydrophobic tunnel. The heterologous expression of the ORDs of human ORP3 or OSBP1 rescued the lethality of seven ORP (yeast OSH1-OSH7) knockout in yeast. In contrast, the PI(4)P-binding site mutant of ORP3 did not complement the OSH knockout cells. The N-terminal PH domain and FFAT motif of ORP3 are involved in protein targeting but are not essential in yeast complementation. This observation suggests that the essential function conserved in the ORPs of yeast and human is mediated by PI(4)P-binding of the ORD domain. This study suggests that the non-vesicular PI(4)P transport is a conserved function of all ORPs in eukaryotes.

Coordination of inter-organelle communication and lipid fluxes by OSBP-related proteins

“…2). Of note, the structural analyses of yeast Osh3p and mammalian ORP3 showed that the lipid-binding cavity of these ORDs is too narrow to accommodate sterols [39,44], consistent with the view that phosphoinositide rather than sterol binding is a common denominator of the ORPs.…”

“…Since the initial Osh4p structure, high-resolution structures of the ORDs of yeast Osh1p [38], Osh3p [39] and Osh6p [40,41], as well as of mammalian ORP1 (Fig. 2) [42], ORP2 [43], and ORP3 [44] have been reported. These structural analyses have revealed interesting differences in the mode of ligand binding to distinct ORP family members: The PI(4,5)P2 binding mode in ORP1 and -2 differs from that of PI4P in the other ORDs.…”

“…Moreover, in ORP1 and -2 one of the PI(4,5)P2 acyl chains is within the ORD cavity while the 2 nd chain is curled up at the top of the cavity [42,43]. In contrast, in Osh4p, Osh6p and ORP3 both acyl chains of the bound PI4P are inserted into the ORD ligand cavity [37,40,41,44](Fig. 2).…”

“…However, the group of Y.J. Im, who reported the structure of yeast Osh3p and recently of human ORP3 ORDs, observed that their lipid-binding cavities are too narrow to accommodate the bulky 4-ringed sterol structure [39,44]. At the same time, the group of A.-C. Gavin [40] documented a function of yeast Osh6p and Osh7p in PS transport from the ER to the PM, suggesting that these ORPs bind PS and PI4P as their two ligands and transport them in opposite directions.…”

Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels

“…Yeast Oxysterol homology (Osh) proteins, mammalian OxySterol Binding Protein (OSBP) and Oxysterol-binding Related Proteins (ORP) proteins form another large LTP protein family ( Kentala et al, 2016 ). At least some 22 crystal structures of LTP domains from Osh or ORP proteins have been tallied so far including Osh1 ( Manik et al, 2017 ), Osh3 ( Tong et al, 2013 ), Osh4 ( Im et al, 2005 ; de Saint-Jean et al, 2011 ), Osh6 ( Maeda et al, 2013 ; Moser von Filseck et al, 2015 ), ORP1, ORP2 ( Wang et al, 2019 ) and ORP3 ( Tong et al, 2021 ) bound to different phosphoinositides and/or cholesterol derivatives; these structures reveal the versatility of this particular LTP fold capable of accommodating different types of ligands (e.g., Osh6 binding PS or PI4P and OSBP binding cholesterol or PI4P). A similar versatility can be described for another large class of LTPs including the PRELI and StART/StARkin domains ( Alpy and Tomasetto, 2014 ) ( Figure 3 ) capable of binding a variety of lipids such as PLs (PA or PS) and sterols or ceramides, respectively.…”