Structural basis of human ORP1-Rab7 interaction for the late-endosome and lysosome targeting

Abstract

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of lipid transfer proteins conserved in eukaryotes. ORP1 transports cholesterol at the interface between the late endosomes/lysosomes (LELs) and the endoplasmic reticulum (ER). ORP1 is targeted to the endosomal membranes by forming a tripartite complex with the LE GTPase Rab7 and its effector RILP (Rab7-interacting lysosomal protein). Here, we determined the crystal structure of human ORP1 ANK domain in complex with the GTP-bound form of Rab7. ORP1 ANK binds to the helix α3 of Rab7 located away from the switching regions, which makes the interaction independent of the nucleotide-binding state of Rab7. Thus, the effector-interacting switch regions of Rab7 are accessible for RILP binding, allowing formation of the ORP1-Rab7-RILP complex. ORP1 ANK binds to Rab7 and the Rab7-RILP complex with similar micro-molar affinities, which is consistent with the independence binding of ORP1 and RILP to Rab7. The structural model of the ORP1-Rab7-RILP complex correlates with the recruitment of ORP1 at the LEL-ER interface and the role in lipid transport and regulation.

Fig 1. Association of ORP1 ANK and Rab7.

(A) Schematic representation of the domain structures of human ORP1 and Rab7. The domains with the structures determined in this study are indicated with blue arrows. (B) Size exclusion chromatography (SEC) profiles of ORP1 ANK (5–152), Rab7 (2–195), RILP (241–320) and their mixtures. To analyze the protein-protein interaction, ORP1, Rab7, and RILP were mixed in a 1:1:1 molar ratio and incubated at room temperature for 30 min prior to SEC. Individual ORP1 ANK or Rab7 protein was applied separately as a control. The SDS-PAGE analysis of the major peaks are shown in the right panel. (C) Sequence alignments of the ANK domains from human, mouse, and rat ORP1 homologs. The residues forming the Rab7-binding sites are shaded in red. The variable residues in these species are shaded in dark blue.

Fig 2. Overall structure of human ORP1 ANK and Rab7 complex.

(A) Cylindrical representation of the ORP1-Rab7 complex. The bound GTP and Mg²⁺ ion are shown in spheres. The disordered C-terminus of Rab7 is indicated with a dotted line. Top view of the structure is shown in the right panel. (B) 2.1 Å 2F₀-Fc electron density maps with the final model superimposed. The Rab7 structure is shown in green and ORP1 ANK in blue. (C) Overall structure of human ORP1 ANK domain.

Fig 3. Formation of the ORP1-Rab7-RILP complex.

(A) Binding interface of the ORP1 ANK and Rab7. The residues forming the interface are shown in stick models. The hydrogen bonds are shown in dashed lines. (B) Measurement of binding affinity of ORP1 ANK to Rab7 by isothermal titration calorimetry. The purified Rab7 or Rab7-RILP complex with the concentration of 0.1 mM was titrated with 1 mM of ORP1 ANK. Rab7 was either loaded with GTP or GDP. (C) Overall structure of the ORP1 ANK-Rab7-RILP complex. The structure was constructed by combining the structures of Rab7-RILP (PDB id: 1YHN) and ORP1 -Rab7 (this study) using PyMOL (https://pymol.org). The missing C-terminal residues (186–203) of Rab7 were indicated with dotted lines. The C-terminal prenyl groups which anchor Rab7 onto the endosomal membrane are shown in black lines.

Fig 4. Schematic model of ORP1 function at the LEL-ER membrane contact site.

The prenylated Rab7 localizes to the late-endosomal/lysosomal membrane. GTP-loaded Rab7 associated with the dimeric RILP recruits two molecules of ORP1 by ORP1 ANK-Rab7 interaction. The ORP1 has additional targeting modules such as a PH domain and a FFAT motif which target to the LEL-ER membrane contact sites. The ORD domain of ORP1 might transport PI4P and cholesterol between the membranes.