College of Pharmacy, Chonnam National University, Gwangju, Republic of Korea.
PLoS One. 2021 Apr 15;16(4):e0248781. doi: 10.1371/journal.pone.0248781. eCollection 2021.
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.
人源 ORP3 属于甾醇结合蛋白（OSBP）家族的脂质转运蛋白，参与脂质运输和细胞信号转导。ORP3 定位于内质网-质膜界面，与脂质转运和黏附斑动力学有关。本研究报道了人源 ORP3 的 ORD（OSBP 相关结构域）在apo 形式和与磷脂酰肌醇 4-磷酸结合形式下的 2.6-2.7Å 结构。ORP3 ORD 呈现出一个螺旋夹β-桶结构，具有一个深的疏水性口袋，该口袋在 OSBP 基因家族中是保守的。ORP3 通过隧道入口周围和疏水性口袋中的残基与 PI(4)P 结合，而由于狭窄的疏水性隧道，它缺乏固醇结合。人源 ORP3 或 OSBP1 的 ORD 的异源表达挽救了酵母中七个 ORP（酵母 OSH1-OSH7）敲除的致死性。相比之下，ORP3 的 PI(4)P 结合位点突变体不能补充 OSH 敲除细胞。ORP3 的 N 端 PH 结构域和 FFAT 基序参与蛋白靶向，但在酵母互补中不是必需的。这一观察表明，在酵母和人类的 ORP 中保守的必需功能是由 ORD 结构域与 PI(4)P 的结合介导的。本研究表明，非囊泡 PI(4)P 转运是真核生物中所有 ORP 的保守功能。
