Structural basis of human PDZD8–Rab7 interaction for the ER-late endosome tethering

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.

www.nature.com/scientificreports/ In this study, to identify the structural determinants for the GTP-dependent interaction of PDZD8 and Rab7, we determined the structure of the human PDZD8 CC and Rab7-GTP complex. The PDZD8 CC forms a flat structure of three α-helices interacting with two Rab7 molecules on opposite sides of the coiled-coil. The PDZD8 CC associates with the active Rab7 by recognizing the switch I/II and interswitch regions. Our structural model of the PDZD8 CC-Rab7 complex is consistent with the role of PDZD8 in membrane tethering and lipid transport at the membrane contact sites.

Results
Overall structure of the PDZD8 CC-Rab7 complex. Human PDZD8 contains an N-terminal transmembrane helix (residues 2-27), followed by a lipid-binding SMP domain and a PDZ domain. The C-terminal region of PDZD8 contains a C1 domain (residue 841-891) and a coiled-coil domain (residue 1010-1100). The middle region (residue 449-841) connecting the PDZ and C1 domains is made of a long disordered loop of about 400 amino acids (Fig. 1A). To obtain a structural insight into the LE-ER tethering of PDZD8 by the PDZD8 CC-Rab7 interaction, we performed X-ray crystallographic studies of the PDZD8 CC domain in complex with the Rab7-GTP. The recombinant proteins of the PDZD8 CC and GTP-loaded Rab7 were purified individually. 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 (residue 999-1104) for crystallographic studies. To obtain the crystals of the PDZD8 CC-Rab7 complex, the proteins were mixed in a 1:1 molar ratio and were incubated for one hour prior to crystallization setup. The hexagonal crystals of the PDZD8 CC-Rab7 complex with P6 5 space group appeared in a week. The structure of the PDZD8 CC-Rab7 complex was determined at 2.1 Å resolution by molecular replacement method using a structure of the GTP-bound Rab7 (Table 1). In the asymmetric unit of the crystal, there were two molecules of Rab7 bound to a single molecule of the PDZD8 CC. The electron densities of GTP-ligands in the Rab7 molecules were clearly visible (Fig. 1B). The PDZD8 CC forms a coiled-coil structure within a single polypeptide chain and does not have intermolecular coiled-coil interaction. The PDZD8 CC starts with an N-terminal ten-residue loop followed by three α-helices (Fig. 1C). The first α-helix (residue 1010-1023) is short with 3.8-helical turns. The second and third α-helices form a 55 Å long antiparallel coiled-coil with ten helical turns. The three α-helices form a flat structure arranged in an α1-α3-α2 topology. The three antiparallel helices are held together mainly by hydrophobic interaction with additional three hydrogen-bonds and three salt bridges. The Rab7 molecules make an asymmetric bivalent binding to the single PDZD8 CC domain (Fig. 1D,E). Two Rab7 molecules (Rab7a-A and Rab7a-B) bind on the opposite sides The disordered loops at the C-termini of Rab7 are indicated with dotted lines. The top view of the structure is shown in the right panel. The switch I residues were colored in magenta, and switch II residues in orange. (E) Binding interfaces of Rab7 and the PDZD8 CC. The PDZD8 CC is shown in surface representation with the residues interacting with Rab7 colored in yellow. PDZD8 CC recognizes the conserved hydrophobic triad of Rab7 in the active conformation. The PDZD8 was known to bind Rab7 in a GTP-dependent manner 7,8,10 . The structure of the PDZD8 CC-Rab7 complex reveals the structural determinants of the GTP-dependent interaction. Two Rab7 molecules loaded with GTP have an active closed conformation of switch I and II loops. Rab7-A and Rab7-B have an almost identical structure except for slight conformational variation in the switch I and II loops with the C α rmsd of 1.38 Å for 171 residues ( Fig. 2A). Rab7-A forms two salt bridges and six hydrogen bonds with PDZD8. The PDZD8 CC utilizes four residues of the N-terminal loop and 11 residues of the three α-helices for Rab7-A binding, forming a buried surface area of 386 Å 2 (Fig. 2B). The direct recognition of the PDZD8 CC on the closed conformation of switch loops explains the GTP-dependent interaction of PDZD8 and Rab7. The switch I and II loops of Rab7-A interact with the N-terminal loop and the helix α2/α3 of PDZD8. The N-terminal loop of the PDZD8 CC binds in the cleft between the helix α2 and the switch II loop, inserting L1001 into the hydrophobic pocket (Fig. 2C). Phe45 and Trp62 in the interswitch and Phe77 of switch II make hydrophobic contact with the helix α1. These three conserved aromatic residues adjacent to switch I and II regions were known as the hydrophobic triad, a major structural determinant for effector binding and specificity in Rab proteins 15 . In addition, Ile41 of switch I and Phe70 of switch II interact with the hydrophobic patch (Leu1051 and Leu1084) on the α2-α3 helices of PDZD8. Rab7-B contacts with the 19 residues of the PDZD8 CC, forming a buried surface area of 224 Å 2 (Fig. 2B). The aromatic triad of Rab7-B forms a hydrophobic interaction with the flat surface on the α1, α2, and α3 helices of the PDZD8 CC (Fig. 2D). The α2 and α3 helices of the PDZD8 CC contact Ile41 and Gly42 of the switch I loop and the interswitch residues. The switch II loop of Rab7-B makes a hydrophobic contact with the shallow pocket between the α2 and α3 of the PDZD8 CC. Both Rab7-A and Rab7-B interact www.nature.com/scientificreports/ with PDZD8 using the switch I/II and interswitch regions as commonly observed in the Ras-like small GTPases in complex with their effectors 16 . Even though Rab7-A and Rab7-B bind the opposite sides of the PDZD8 CC, both Rab7 molecules utilize the conserved hydrophobic triad for PDZD8 binding, suggesting a conserved mode of Rab7 effector recognition.

PDZD8 CC-Rab7 interaction is dependent on the GTP-binding state of Rab7.
To analyze the GTP-dependency and stoichiometry of the interaction, we measured the affinity of the various PDZD8 CC and Rab7 constructs by ITC (Fig. 3A,B). To obtain homogeneous nucleotide-binding states of Rab7, we used the constitutively GTP-binding Rab7 Q67L and GDP-binding Rab7 T22N rather than wild-type Rab7 for ITC experiments. The PDZD8 CC constructs spanning residues 980-1123 and 994-1123 interacted with Rab7. However, the PDZD8 CC construct lacking helix α1 (1021-1123) did not bind Rab7. Since the crystal structure displayed the binding of two Rab7 molecules to a single PDZD8 CC molecule, we used a two-site binding model for the wild-type PDZD8 CC and Rab7 interaction. The MBP-PDZD8 CC bound two GTP-loaded Rab7 molecules with Kd values of 0.67 and 11.1 μM, respectively. The primary binding site displayed 16 times higher binding affinity than the secondary site. We interpreted that the high binding affinity represents the Rab7-A interaction considering the 1.7 times larger interface area of Rab7-A than the area of Rab7-B. To confirm the 2:1 binding mode observed in the crystal structure, we constructed two Rab7-binding site mutants of the PDZD8 CC.
Ala1086 of PDZD8 is located in the center of the binding interface between PDZD8 and Rab7-B. Therefore, A1086R mutation was expected to interfere with the Rab7-B binding without affecting the interaction with Rab7-A (Fig. 2D). Ile1092 is located in the center of PDZD8-Rab7-A interface (Fig. 2C). The I1092E mutation was expected to disrupt the Rab7-A binding. As predicted from the structure analysis, PDZD8 A1086R retained the high-affinity binding with a Kd of 0.66 μM when analyzed by a single-site binding model. However, PDZD8 I1092E mutant displayed a Kd of 9.2 μM, interfering with the high-affinity binding of Rab7-A. Consistently, when both Rab7-A and Rab7-B binding sites were altered with A1086R/I1092E mutations, the affinity of PDZD8 to Rab7 was lost completely. These observations confirm that the PDZD8 CC has two Rab7 binding sites with

Conservation of a binding mode in Rab7-effector complexes. Structural comparison of the various
Rab7-effector complexes suggests that the GTP-dependent interaction has a conserved mode of recognition on the active conformation of Rab7 (Fig. 4A). The hydrophobic triad on switch II and interswitch regions was first identified in Rab proteins and was proposed to be a major structural determinant for GTP-dependent effector binding and specificity 15 . So far, PDZD8 7 , RILP 18 , Rubicon 19 , and ORP1L 20 were identified to be the Rab7 effector proteins with their structures reported. The PDZD8 CC, RILP CC, and Rubicon RH domains commonly recognize the conserved hydrophobic triad in the switch II and interswitch regions of Rab7 14,21 . However, the ORP1L ANK domain binds on the surface distal to the switch I/II regions, explaining the nucleotide-independent interaction of ORP1L 22,23 . The Rab7-binding domain of RILP is a coiled-coil homodimer and interacts with two www.nature.com/scientificreports/ Rab7-GTP molecules, forming a symmetric dyad complex 14 . The RILP-Rab7 complex exposes the C-termini of two Rab7 molecules in the same direction toward membranes. Although the PDZD8 CC does not form a coiled-coil dimer, two Rab7 molecules form a similar dyad configuration around the central PDZD8 CC monomer (Fig. 4A). Collectively, despite no structural and sequence similarities of the GTP-dependent Rab7 effector proteins, they share a common mode of Rab7 recognition.
Structural model of the PDZD8 CC-Rab7 complex on the membrane. The structural model of the membrane-bound PDZD8 CC-Rab7 complex demonstrates the spatial arrangement of the subunits required for membrane recruitment (Fig. 4B). PDZD8 resides on the ER membrane by its N-terminal transmembrane domain and is targeted to the LEL membrane by its C-terminal coiled-coil domain via interaction with Rab7 7 . Rab7 is anchored to the LEL membrane via prenylation of the two cysteine residues in the C-terminus. Rab7 presumably lies far away due to their long flexible tails that precede the prenylation sites. The C-terminal cysteine residue is connected to the Rab7 core by a flexible 30 residue loop, which might allow the binding of two membrane-anchored Rab7 molecules to the PDZD8 CC in different orientations. We modeled a structure of the PDZD8 CC-Rab7 complex on the membrane considering the orientation of the Rab7 C-termini (Fig. 4B). The Rab7-binding domain of PDZD8 forms a flat coiled-coil structure, allowing binding of two Rab7 molecules simultaneously on the opposite sides of the PDZD8 CC. The asymmetric bivalent binding of Rab7 and the membrane anchoring by Rab7 impose a constraint in the orientation of the PDZD8 CC-Rab7 complex, positioning the coiled-coil domain parallel to the membrane surface (Fig. 4B). The switch I/II and interswitch regions of Rab7 contact with PDZD8, which makes the interaction GTP-dependent. The divalent binding of Rab7 effectors may help efficient tethering between the membrane contact sites by increasing the avidity of the interaction. The presence of the ER-targeting transmembrane helix and the Rab7-binding CC is consistent with the role of PDZD8 in membrane tethering and lipid transport at the membrane contact sites (Fig. 4C). www.nature.com/scientificreports/

Discussion
We investigated the structural basis of specific recognition of the GTP-loaded Rab7 by the PDZD8 CC. PDZD8 is an LTP tethered at the ER-endo/lysosome interface and contributes to endosome maturation in cooperation with Protrudin 9,10 . Recruitment of PDZD8 to the MCSs is mediated by the N-terminal TM region and the C-terminal Rab7-binding domain, which is crucial for its lipid transport function 7 . The bivalent Rab binding to effectors is common in many Rab GTPases 16 . The RBDs commonly adopt a coiled-coil dimer and bind to two Rab GTPases on the opposite sides of the central coiled-coil. Symmetric association of Rab GTPases to the coiled-coil dimer of the effectors were observed for the Rab5:Rabaptin5 24 , Rab6:GCC185 25 , Rab6:KIF20A 26 , Rab7:RILP 14 , Rab11:FIP2 27 and Rab11:FIP3 28,29 complexes. The bivalent Rab binding to effectors was suggested to increase the effector residence time at the membrane. It also imposes a restricted orientation to the effectors, allowing the effectors to project into the cytosol to perform their function 16 . Unlike the two-fold symmetry of typical Rab:effector complexes, a single molecule of the PDZD8 CC makes an asymmetric binding to two Rab7 molecules. The bivalent binding of PDZD8 to Rab7 seems to be advantageous in increasing the avidity of the interaction, which might provide stable tethering between the ER and LEs. PDZD8 associates with an ER transmembrane protein Protrudin and regulates endosome trafficking through interaction with the GTP-bound form of Rab7 8,9,12 . The tripartite complex of PDZD8-Rab7-Protrudin is known to regulate the plus-end-directed movement of endosomes for the recycling and secretion of cargos. In contrast, the ORP1L-Rab7-RILP complex regulates the minus end-directed motility of endosomes for lysosomal degradation 16 . ORP1L is a cholesterol sensor/transporter which is recruited to the ER-LE/lysosome contact sites by association with Rab7 20,22 . Rab7 cannot bind PDZD8 and RILP/ORP1L simultaneously due to their overlapping binding sites, correlating with their role in the opposite directions of endosomal transport. Maturing endosomes undergo changes in the lipid composition, such as the depletion of phosphatidylserine and the enrichment of lysobisphosphatidic acid 30,31 . It implies that the direction of endosome sorting and endosome maturation might be controlled by associating with the specific Rab7-effectors including the lipid transfer proteins, PDZD8 and ORPL1.
In conclusion, this study demonstrates the key structural determinants for the GTP-dependent recruitment of PDZD8 to the LEs by Rab7-GTP. The bivalent Rab7-binding to PDZD8 might be advantageous for the stable tethering at the MCSs by providing avidities. PDZD8 recruited to the ER-LE interfaces by Rab7 might allow efficient lipid transport by its SMP domain for the late endosome/lysosome processing.

Materials and methods
Cloning of human PDZD8 CC and Rab7. The full-length clones for human PDZD8 (hMU002500, Uni-Prot id: Q8NEN9) and Rab7 (hMU001574, UniProt id: P51149) were purchased from 21C Frontier Human Gene Bank (KRIBB, Daejeon, Republic of Korea). DNA encoding the C-terminal coiled-coil (CC) domain (residues 994-1123) of human PDZD8 was amplified by polymerase chain reaction and subcloned into the NcoI/ XhoI site of a modified pHIS2 vector 32 . The PDZD8 CC was tagged with the N-terminal hexahistidines followed by a thrombin protease cleavage site (LVPR/GS). The DNA encoding human Rab7 (residue 2-195) was subcloned into the BamHI/XhoI site of the modified pHIS-2 vector. The dominant active GTP-binding (Q67L) and dominant-negative GDP-binding (T22N) mutants of Rab7 were prepared by point mutagenesis. Escherichia coli strain BL21(DE3) cells transformed with the plasmid encoding the PDZD8 CC or Rab7 were grown to an OD 600 of 0.8 at 37 °C in LB medium. Cells were induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM and were incubated for 12 h overnight at 20 °C prior to harvesting.
Protein expression and purification. Cells expressing the PDZD8 CC were resuspended in 2X PBS buffer containing 20 mM imidazole (lysis buffer) and lysed by sonication. The supernatant containing the Histagged PDZD8 CC was loaded to a Ni-NTA affinity column. The Ni-NTA column was thoroughly washed with the lysis buffer. The target protein was eluted from the column using a buffer containing 100 mM Tris-HCl pH 8.0 (final), 300 mM imidazole. The eluate was concentrated to 10 mg/ml using Amicon Ultra-15 centrifugal filter. The His-tag was cleaved by 10 international unit (IU) of thrombin protease (Reyon Pharmaceutical) per 10 mg of recombinant protein. The cleaved sample was subjected to size-exclusion chromatography on a HiLoad Superdex 200 column equilibrated with 20 mM Tris-HCl pH 8.0, 150 mM NaCl. The fractions containing the PDZD8 CC were concentrated by the centrifugal filter to 10 mg/ml for crystallization. The Rab7 Q67L or T22N mutant were purified by the same procedure as the PDZD8 CC. To inhibit GTP hydrolysis of the PDZD8-Rab7 complex required for crystallization, we used a dominant active Q67L mutant of Rab7. The purified Rab7 Q67L was supplemented with the final 1 mM of GTP.
Crystallization and crystallographic analysis. To obtain the crystals of the PDZD8 CC-Rab7 complex, we mixed the purified PDZD8 CC and Rab7 proteins with a 1:1 molar ratio at a total 10 mg/ml, and incubated for 1 h at room temperature before crystallization setup. Preliminary crystallization experiments were carried out at 22 °C in 96-well crystallization plates using customized crystallization screening solutions by dispensing 0. 8