Structural basis of human PDZD8\u2013Rab7 interaction for the ER-late endosome tethering

Haider Khan,Young Jun Im,Lin Chen,Lingchen Tan

doi:10.1038/s41598-021-98419-5

Abstract

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation.

Highlights

The lipid compositions of membranes are unique for each subcellular organelle in eukaryotes, and the proper distribution of lipids is crucial for the organellar function[1]
We tested the Rab7-binding activity of various PDZD8 CC constructs with different domain boundaries by isothermal titration calorimetry (ITC) and selected the smallest construct for crystallographic studies
To obtain the crystals of the PDZD8 CC–Rab[7] complex, the proteins were mixed in a 1:1 molar ratio and were incubated for one hour prior to crystallization setup

Summary

Introduction

The lipid compositions of membranes are unique for each subcellular organelle in eukaryotes, and the proper distribution of lipids is crucial for the organellar function[1]. Rab[7] recruits mainly through the switch regions, structurally diverse effector proteins such as PDZD8, FYCO1, RILP, and Rubicon[5]. PDZD8 associates with an ER protein Protrudin and coordinates endocytic flow with lipid transport at the ER–endosome/lysosome interface[8,9,10]. The Rab-binding domains (RBDs) of PDZD8 and RILP bind only to the active form of R ab[77,14]. The PDZD8 CC forms a flat structure of three α-helices interacting with two Rab[7] molecules on opposite sides of the coiled-coil. Our structural model of the PDZD8 CC–Rab[7] complex is consistent with the role of PDZD8 in membrane tethering and lipid transport at the membrane contact sites

Methods

Results

Conclusion