Structural basis for activation of fungal sterol receptor Upc2 and azole resistance.

Fungal transcription factor Upc2 senses ergosterol levels and regulates sterol biosynthesis and uptake. Constitutive activation of Upc2 causes azole resistance in Candida species. We determined the structure of ergosterol-bound Upc2, revealing the ligand specificity and transcriptional regulation. Ergosterol binding involves conformational changes of the ligand-binding domain, creating a shape-complementary hydrophobic pocket. The conserved helix α12 and glycine-rich loop are critical for sterol recognition by forming the pocket wall. The mutations of the glycine-rich loop inhibit ligand binding by steric clashes and constitutively activate Upc2. The translocation of Upc2 is regulated by Hsp90 chaperone in a sterol-dependent manner. Ergosterol-bound Upc2 associates with Hsp90 using the C-terminal tail, which retains the inactive Upc2 in the cytosol. Ergosterol dissociation induces a conformational change of the C-terminal tail, releasing Upc2 from Hsp90 for nuclear transport by importin α. The understanding of the regulatory mechanism provides an antifungal target for the treatment of azole-resistant Candida infections.

Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex.

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

Activation of JNK and p38 in MCF-7 Cells and the In Vitro Anticancer Activity of Alnus hirsuta Extract.

JNK and p38 are important mitogen-activated protein kinases (MAPKs) that respond to stress stimuli. The stress-activated MAPKs associated with apoptotic cell death play vital roles in mammalian cells. Alnus hirsuta, which contains abundant diarylheptanoids derivatives, is a valuable medicinal plant. The CHCl3 extract (AHC) containing platyphyllenone (1) and platyphyllone (3) as main compounds showed in vitro anticancer effects. We report the biological activities of A. hirsuta extract associated with the regulation of apoptosis and JNK and p38 in MCF-7 breast cancer cells. Levels of phospho-JNK and phospho-p38 by AHC treatment were evaluated by enzyme-linked immunosorbent assay (ELISA). ROS production, apoptotic effect, and DNA contents of the cells were measured by flow cytometry. The two diarylheptanoids 1 and 3 and the AHC extract exhibited cytotoxic effects on MCF-7 cells in MTT assay, with IC50 values of 18.1, 46.9, 260.0 µg/mL, respectively. AHC induced ROS generation and elevated the endogenous levels of phospho-JNK and phospho-p38. AHC resulted in apoptosis and cell cycle arrest. We suggest that the antitumor effect of A. hirsuta extract is achieved by apoptosis promotion and cell cycle arrest mediated by the activation of JNK and p38 signaling pathway via ROS generation.

Structural analysis of human sterol transfer protein STARD4.

The steroidogenic acute regulatory protein (StAR)-related lipid transfer domain-4 (STARD4) is a sterol-binding protein that is involved in cholesterol homeostasis by intracellular sterol transport. In this work, we determined the crystal structures of human STARD4 and its Ω1-loop mutant in apo forms at 1.95 and 1.7 Šresolutions, respectively. The structure of human STARD4 displays a conserved α-helix/ß-grip fold containing a deep hydrophobic pocket. The Ω1-loop which serves as a lid for the hydrophobic pocket has a closed conformation. The shape of the sterol-binding cavity in the closed form is not complementary to accommodate cholesterol, suggesting that a conformational change of the Ω1-loop is essential for sterol binding. The human STARD4 displayed sterol transfer activity between liposomes, and the mutations in the Ω1-loop and the hydrophobic wall abolished the transfer activity. This study confirms the structural conservation of the STARD4 subfamily proteins and the flexibility of the Ω1-loop and helix α4 required for sterol transport.

Structural insights into nonvesicular lipid transport by the oxysterol binding protein homologue family.

Sterols such as cholesterol in mammals and ergosterol in fungi are essential membrane components and play a key role in membrane function and in cell signaling. The intracellular distribution and processing of sterols and other phospholipids are in part carried out by oxysterol binding protein-related proteins (ORPs) in eukaryotes. Seven ORPs (Osh1-Osh7 proteins) in yeast have distinct functions in maintaining distribution, metabolism and signaling of intracellular lipids but they share at least one essential function. Significant progress has been made in understanding the ligand specificity and mechanism of non-vesicular lipid transport by ORPs. The unique structural features of Osh proteins explain the diversity and specificity of functions in PI(4)P-coupled lipid transport optimized in membrane contact sites. This review discusses the current advances in structural biology regarding this protein family and its potential functions, introducing them as the key players in the novel pathways of phosphoinositide-coupled directional transport of various lipids. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Resistance Mechanisms and Clinical Features of Fluconazole-Nonsusceptible Candida tropicalis Isolates Compared with Fluconazole-Less-Susceptible Isolates.

We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 µg/ml), 12 FLS (MIC, 1 to 2 µg/ml), and 14 control (MIC, 0.125 to 0.5 µg/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P = 0.004), but there were no differences in those of CDR1 or MDR1 Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P = 0.011) and immunosuppression (6/6 versus 3/10; P = 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression.

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus.

Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank-Homer complexes by protein-protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein-protein interaction module for the synaptic protein clustering.

Structural basis of the activation of MARTX cysteine protease domain from Vibrio vulnificus.

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and ß-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded ß-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the ß-flap inducing the active conformation of catalytic residues. The ß-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δß-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the ß-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the ß-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.

Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation.

Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane-tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding mu homology domains (muHDs). In vitro and in vivo assays confirmed membrane-tubulation activity for muniscin EFC/F-BAR domains. The muHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane-tubulation activity that is important for regulating endocytosis.

Crystallization and preliminary X-ray crystallographic analysis of sterol transcription factor Upc2 from Saccharomyces cerevisiae.

Upc2, a zinc-cluster transcription factor, is a regulator of ergosterol biosynthesis in yeast. In response to sterol levels, the transcriptional activity of Upc2 is controlled by the C-terminal domain. In this study, the C-terminal regulatory domain of Upc2 from Saccharomyces cerevisiae was purified and crystallized by the vapour-diffusion method. To improve the diffraction quality of Upc2 crystals, a Upc2 fusion protein in which 11 residues of the variable loop (residues 715-725) were replaced by T4 lysozymes in Upc2 (Upc2-T4L) was engineered. The Upc2-T4L crystals diffracted to 2.9 Å resolution using synchrotron radiation. The crystal was trigonal, belonging to space group P3(2) with unit-cell parameters a = 67.2, b = 67.2, c = 257.5 Å. The Matthews coefficient was determined to be 3.41 Å(3) Da(-1) with two molecules in the asymmetric unit. Initial attempts to solve the structure by the single-anomalous dispersion technique using selenomethionine were successful.

Microtubes with gradient decellularized porcine sciatic nerve matrix from microfluidics for sciatic nerve regeneration.

Long-range peripheral nerve defect is a severe and worldwide disease. With the increasing development of tissue engineering, the excellent ability of nerve extracellular matrix (ECM) in peripheral nerve injury (PNI) has been widely studied and verified. Here, we present a novel microtube that contains gradient decellularized porcine sciatic nerve ECM hydrogel (pDScNM-gel) from microfluidics for sciatic nerve regeneration. The pDScNM is confirmed to enhance cell proliferation and migration, and improve the axon growth of primary dorsal root ganglions (DRGs) in a concentration-related manner. These behaviors were also achieved when cells were co-cultured in a gradient pDScNM microtube. The in vivo sciatic nerve regeneration and functional recovery were also demonstrated by assembling the gradient pDScNM microtubes with a medical silicon tube. These results indicated that the microtubes with gradient pDScNM could act as a promising alternative for repairing peripheral nerve defects and showed great potential in clinical use.

Direct inhibition of human and rat 11ß-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis.

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11ß-HSD2. C8-C14 PFAS at 100 µM significantly inhibited human 11ß-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 µM) > C11 (15.09 µM) > C12 (18.43 µM) > C9 (20.93 µM) > C13 (124 µM) > C14 (147.3 µM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11ß-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11ß-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11ß-HSD2 but not rat 11ß-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11ß-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11ß-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11ß-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11ß-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11ß-HSD2.

rFGF4 alleviates lipopolysaccharide-induced acute lung injury by inhibiting the TLR4/NF-κB signaling pathway.

Acute lung injury (ALI) is a serious and common clinical disease. Despite significant progress in ALI treatment, the morbidity and mortality rates remain high. However, no effective drug has been discovered for ALI. FGF4, a member of the FGF family, plays an important role in the regulation of various physiological and pathological processes. Therefore, in the present study, we aimed to study the protective effects of FGF4 against LPS-induced lung injury in vivo and in vitro. We found that rFGF4 treatment improved the lung W/D weight ratio, the survival rate, immune cell infiltration and protein concentrations in mice with LPS-induced ALI. Histological analysis revealed that rFGF4 significantly attenuated lung tissue injury and cell apoptosis. Furthermore, rFGF4 inhibited the activation of the TLR4/NF-κB signaling pathway and the production of pro-inflammatory mediators in LPS-injured lung tissues, murine alveolar macrophages (MH-S) and murine pulmonary epithelial (MLE-12) cells. The results of cell experiments further verified that rFGF4 inhibited the production of inflammatory mediators in MH-S cells and MLE-12 cells by regulating the TLR4/NF-κB signaling pathway. These results revealed that rFGF4 protected lung tissues and inhibited inflammatory mediators in mice with LPS-induced ALI by inhibiting the TLR4/NF-κB signaling pathway in MH-S and MLE-12 cells.

Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae.

Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum-plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2.3 Šresolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=98.03, b=91.31, c=84.13 Å, ß=81.41°. With two molecules in the asymmetric unit, the Matthews coefficient was 3.13 Å3 Da(-1). Initial attempts to solve the structure by molecular-replacement techniques using T4 lysozyme as a search model were successful. The C-terminal OSBP-related domain (OBD) of Osh3 was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 1.5 Šresolution. The crystal was orthorhombic, belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=41.57, b=87.52, c=100.58 Å. With one molecule in the asymmetric unit, the Matthews coefficient was 2.01 Å3 Da(-1). Initial attempts to solve the structure by the single-wavelength anomalous dispersion technique using bromine were successful.

Structural basis of ligand recognition and transport by Sfh2, a yeast phosphatidylinositol transfer protein of the Sec14 superfamily.

Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting phosphatidylinositol (PI) and a secondary ligand between the organellar membranes in eukaryotes. Yeast Sfh2 is a PITP that transfers PI and squalene without phosphatidylcholine transfer activity. To investigate the structural determinants for ligand specificity and transport in Sfh2, crystal structures of Sfh2 in complex with PI and squalene were determined at 1.5 and 2.4 Šresolution, respectively. The inositol head group of PI is recognized by highly conserved residues around the pocket entrance. The acyl chains of PI bind into a large hydrophobic cavity. Squalene is accommodated in the bottom of the cavity entirely by hydrophobic interactions. The binding of PI and squalene are mutually exclusive due to their overlapping binding sites, correlating with the role in lipid exchange. The binding mode of PI is well conserved in Sfh family proteins. However, squalene binding is unique to the Sfh2 homolog due to the specific hydrophobic residues forming a shape-complementary binding pocket. Recombinant apo Sfh2 forms a homodimer in vitro by the hydrophobic interaction of the gating α10-α11 helices in an open conformation. Ligand binding closes the lid and dissociates the dimer into monomers. This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2.

Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides.

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)-related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.

Structural mechanism for sterol sensing and transport by OSBP-related proteins.

The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans, and are implicated in the regulation of sterol homeostasis and in signal transduction pathways. Here we report the structure of the full-length yeast ORP Osh4 (also known as Kes1) at 1.5-1.9 A resolution in complexes with ergosterol, cholesterol, and 7-, 20- and 25-hydroxycholesterol. We find that a single sterol molecule binds within a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible amino-terminal lid and surrounded by basic residues that are critical for Osh4 function. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 A resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favouring transport across aqueous barriers and signal transmission. The structure of Osh4 in the absence of ligand exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. On the basis of these observations, we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signalling cycle.

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

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 ß-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.

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.