Structural basis of hydroxycarboxylic acid receptor signaling mechanisms through ligand binding.

Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only limited information is available, however, on the active-state binding of HCAs with agonists. Here, we present cryo-EM structures of human HCA2-Gi and HCA3-Gi signaling complexes binding with multiple compounds bound. Agonists were revealed to form a salt bridge with arginine, which is conserved in the HCA family, to activate these receptors. Extracellular regions of the receptors form a lid-like structure that covers the ligand-binding pocket. Although transmembrane (TM) 6 in HCAs undergoes dynamic conformational changes, ligands do not directly interact with amino acids in TM6, suggesting that indirect signaling induces a slight shift in TM6 to activate Gi proteins. Structural analyses of agonist-bound HCA2 and HCA3 together with mutagenesis and molecular dynamics simulation provide molecular insights into HCA ligand recognition and activation mechanisms.

Structural analysis of the water channel AQP2 by single-particle cryo-EM.

Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful technique routinely used to determine the structures of membrane proteins. Because the single-particle method enables structural analysis of a whole protein with flexible parts that interfere with crystallization, we have focused our efforts on analyzing water channel structures. Here, utilizing this system, we analyzed the structure of full-length aquaporin-2 (AQP2), a primary regulator of vasopressin-dependent reabsorption of water at the renal collecting ducts. The 2.9 Å resolution map revealed a cytoplasmic extension of the cryo-EM density that was presumed to be the highly flexible C-terminus at which the localization of AQP2 is regulated in the renal collecting duct cells. We also observed a continuous density along the common water pathway inside the channel pore and lipid-like molecules at the membrane interface. Observations of these constructions in the AQP2 structure analyzed without any fiducial markers (e.g., a rigidly bound antibody) indicate that single-particle cryo-EM will be useful for investigating water channels in native states as well as in complexes with chemical compounds.

Recognition Mechanism of a Novel Gabapentinoid Drug, Mirogabalin, for Recombinant Human α2δ1, a Voltage-Gated Calcium Channel Subunit.

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2δ1. Here, to reveal the mirogabalin recognition mechanisms of α2δ1, we present structures of recombinant human α2δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α2δ1 to those of the α2δ2, α2δ3, and α2δ4 isoforms, of which α2δ3 and α2δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α2δ1 ligand recognition.

Development of a deep learning-based method to identify "good" regions of a cryo-electron microscopy grid.

Recent advances in cryo-electron microscopy (cryo-EM) have enabled protein structure determination at atomic resolutions. Cryo-EM specimens are prepared by rapidly freezing a protein solution on a metal grid coated with a holey carbon film; this results in the formation of an ice film on each hole. The thickness of the ice film is a critical factor for high-resolution structure determination; ice that is too thick degrades the contrast of the protein image while ice that is too thin excludes the protein from the hole or denatures the protein. Therefore, trained researchers need to manually select "good" regions with appropriate ice thicknesses for imaging. To reduce the time spent on such tasks, we developed a deep learning program consisting of a "detector" and a "classifier" to identify good regions from low-magnification EM images. In our method, the holes in a low-magnification EM image are detected via a detector, and the ice image on each hole is classified as either good or bad via a classifier. The detector detected more than 95% of the holes regardless of the type of samples. The classifier was trained for different types of samples because the appropriate ice thickness varies between sample types. The accuracies of the classifiers were 93.8% for a soluble protein sample (ß-galactosidase) and 95.3% for a membrane protein sample (bovine heart cytochrome c oxidase). In addition, we found that a training data set containing ~ 2100 hole images from 300 low-magnification EM images was sufficient to obtain good accuracy, such as higher than 90%. We expect that the throughput of the cryo-EM data collection step will be greatly improved by using our method.

Morphologic determinant of tight junctions revealed by claudin-3 structures.

Tight junction is a cell adhesion apparatus functioning as barrier and/or channel in the paracellular spaces of epithelia. Claudin is the major component of tight junction and polymerizes to form tight junction strands with various morphologies that may correlate with their functions. Here we present the crystal structure of mammalian claudin-3 at 3.6 Å resolution. The third transmembrane helix of claudin-3 is clearly bent compared with that of other subtypes. Structural analysis of additional two mutants with a single mutation representing other subtypes in the third helix indicates that this helix takes a bent or straight structure depending on the residue. The presence or absence of the helix bending changes the positions of residues related to claudin-claudin interactions and affects the morphology and adhesiveness of the tight junction strands. These results evoke a model for tight junction strand formation with different morphologies - straight or curvy strands - observed in native epithelia.

TDP-43 stabilises the processing intermediates of mitochondrial transcripts.

The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43-related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

RNA-seq analysis of the response of the halophyte, Mesembryanthemum crystallinum (ice plant) to high salinity.

Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.

Tight junctions. Structural insight into tight junction disassembly by Clostridium perfringens enterotoxin.

The C-terminal region of Clostridium perfringens enterotoxin (C-CPE) can bind to specific claudins, resulting in the disintegration of tight junctions (TJs) and an increase in the paracellular permeability across epithelial cell sheets. Here we present the structure of mammalian claudin-19 in complex with C-CPE at 3.7 Å resolution. The structure shows that C-CPE forms extensive hydrophobic and hydrophilic interactions with the two extracellular segments of claudin-19. The claudin-19/C-CPE complex shows no density of a short extracellular helix that is critical for claudins to assemble into TJ strands. The helix displacement may thus underlie C-CPE-mediated disassembly of TJs.

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state.

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

Oligomeric structure and functional characterization of Caenorhabditis elegans Innexin-6 gap junction protein.

Innexin is the molecular component of invertebrate gap junctions. Here we successfully expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels and characterized the molecular dimensions and channel permeability using electron microscopy (EM) and microinjection of fluorescent dye tracers, respectively. Negative staining and thin-section EM of isolated INX-6 gap junction membranes revealed a loosely packed hexagonal lattice and a greater cross-sectional width than that of connexin26 and connexin43 (Cx43)-GFP. In gel filtration analysis, the elution profile of purified INX-6 channels in dodecyl maltoside solution exhibited a peak at ∼400 kDa that was shifted to ∼800 kDa in octyl glucose neopentyl glycol. We also obtained the class averages of purified INX-6 channels from these peak fractions by single particle analysis. The class average from the ∼800-kDa fraction showed features of the junction form with a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class averages from the ∼400-kDa fraction showed diameters of up to 140 Å in the presence of detergent micelles. These findings indicate that the purified INX-6 channels are predominantly hemichannels in dodecyl maltoside and docked junction channels in octyl glucose neopentyl glycol. Dye transfer experiments revealed that the INX-6-GFP-His channels are permeable to 3- and 10-kDa tracers, whereas no significant amounts of these tracers passed through the Cx43-GFP channels. Based on these findings, INX-6 channels have a larger overall structure and greater permeability than connexin channels.

Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice.

BACKGROUND: Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. RESULTS: The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. CONCLUSIONS: These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.

Electron tomographic analysis of gap junctions in lateral giant fibers of crayfish.

Innexin-gap junctions in crayfish lateral giant fibers (LGFs) have an important role in escape behavior as a key component of rapid signal transduction. Knowledge of the structure and function of characteristic vesicles on the both sides of the gap junction, however, is limited. We used electron tomography to analyze the three-dimensional structure of crayfish gap junctions and gap junctional vesicles (GJVs). Tomographic analyses showed that some vesicles were anchored to innexons and almost all vesicles were connected by thin filaments. High densities inside the GJVs and projecting densities on the GJV membranes were observed in fixed and stained samples. Because the densities inside synaptic vesicles were dependent on the fixative conditions, different fixative conditions were used to elucidate the molecules included in the GJVs. The projecting densities on the GJVs were studied by immunoelectron microscopy with anti-vesicular monoamine transporter (anti-VMAT) and anti-vesicular nucleotide transporter (anti-VNUT) antibodies. Some of the projecting densities were labeled by anti-VNUT, but not anti-VMAT. Three-dimensional analyses of GJVs and excitatory chemical synaptic vesicles (CSVs) revealed clear differences in their sizes and central densities. Furthermore, the imaging data obtained under different fixative conditions and the immunolabeling results, in which GJVs were positively labeled for anti-VNUT but excitatory CSVs were not, support our model that GJVs contain nucleotides and excitatory CSVs do not. We propose a model in which characteristic GJVs containing nucleotides play an important role in the signal processing in gap junctions of crayfish LGFs.

Human umbilical cord-derived mesenchymal stromal cells differentiate into functional Schwann cells that sustain peripheral nerve regeneration.

Human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) that are available from cell banks can be induced to differentiate into various cell types, thereby making them practical potential sources for cell-based therapies. In injured peripheral nerves, Schwann cells (SCs) contribute to functional recovery by supporting axonal regeneration and myelin reconstruction. Here, we first demonstrate a system to induce UC-MSCs to differentiate into cells with SC properties (UC-SCs) by treatment with ß-mercaptoethanol followed by retinoic acid and a set of specific cytokines. The UC-SCs are morphologically similar to SCs and express SC markers, including P0, as assessed by immunocytochemistry and reverse transcription polymerase chain reaction. Transplantation of UC-SCs into transected sciatic nerves in adult rats enhanced nerve regeneration. The effectiveness of UC-SCs for axonal regeneration was comparable to that of authentic human SCs based on histological criteria and functional recovery. Immunohistochemistry and immunoelectron microscopy also demonstrated myelination of regenerated axons by UC-SCs. These findings indicate that cells with SC properties and with the ability to support axonal regeneration and reconstruct myelin can be successfully induced from UC-MSCs to promote functional recovery after peripheral nerve injury. This system may be applicable for the development of cell-based therapies.

Unique multipotent cells in adult human mesenchymal cell populations.

We found adult human stem cells that can generate, from a single cell, cells with the characteristics of the three germ layers. The cells are stress-tolerant and can be isolated from cultured skin fibroblasts or bone marrow stromal cells, or directly from bone marrow aspirates. These cells can self-renew; form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency; and can differentiate into endodermal, ectodermal, and mesodermal cells both in vitro and in vivo. When transplanted into immunodeficient mice by local or i.v. injection, the cells integrated into damaged skin, muscle, or liver and differentiated into cytokeratin 14-, dystrophin-, or albumin-positive cells in the respective tissues. Furthermore, they can be efficiently isolated as SSEA-3(+) cells. Unlike authentic ES cells, their proliferation activity is not very high and they do not form teratomas in immunodeficient mouse testes. Thus, nontumorigenic stem cells with the ability to generate the multiple cell types of the three germ layers can be obtained through easily accessible adult human mesenchymal cells without introducing exogenous genes. These unique cells will be beneficial for cell-based therapy and biomedical research.

Mechanism of aquaporin-4's fast and highly selective water conduction and proton exclusion.

Members of the aquaporin (AQP) family are expressed in almost every organism, including 13 homologues in humans. Based on the electron crystallographic structure of AQP1, the hydrogen-bond isolation mechanism was proposed to explain why AQPs are impermeable to protons despite their very fast water conduction. The mechanism by which AQPs exclude protons remained controversial, however. Here we present the structure of AQP4 at 2.8 A resolution obtained by electron crystallography of double-layered two-dimensional crystals. The resolution has been improved from the previous 3.2 A, with accompanying improvement in data quality resulting in the ability to identify individual water molecules. Our structure of AQP4, the predominant water channel in the brain, reveals eight water molecules in the channel. The arrangement of the waters provides support for the hydrogen-bond isolation mechanism. Our AQP4 structure also visualizes five lipids, showing that direct interactions of the extracellular surface of AQP4 with three lipids in the adjoining membrane help stabilize the membrane junction.

Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release.

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex-mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)-like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

Formation of aquaporin-4 arrays is inhibited by palmitoylation of N-terminal cysteine residues.

Tetramers of the mammalian water channel aquaporin-4 (AQP4) assemble into square arrays and mediate bidirectional water transport across the blood-brain interface. The aqp4 gene expresses two splicing isoforms. Only the shorter AQP4M23 isoform assembles into square arrays, while the longer AQP4M1 isoform interferes with array formation, presumably due to the additional 22 N-terminal residues. To understand why the N-terminus of AQP4M1 interferes with array formation, we constructed a series of N-terminal deletion mutants and examined their ability to form square arrays in Chinese hamster ovary (CHO) cells using SDS-digested freeze fracture replica labeling. Mutants with deletions of less than seventeen N-terminal residues did not form square arrays and showed dispersed immunogold labels against AQP4 molecules, whereas more deletions led to the formation of square arrays labeled with immunogolds. Furthermore, mutagenic substitution of the two cysteine residues at the position 13 and 17 in the N-terminus of AQP4M1 also resulted in the square array formation. Biochemical analysis and metabolic labeling of transfected CHO cells revealed that the two N-terminal cysteines of AQP4M1 are palmitoylated. These results suggest that palmitoylation of the N-terminal cysteines is the reason for the inability of AQP4M1 to form square arrays.

Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8.

Expression of the human Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8, in E. coli and the substrate specificity of the expressed protein were examined. The tissue distribution of mRNA expression of the human Ke 6 gene was also studied using real-time PCR. Human Ke 6 gene was expressed as an enzymatically-active His-tag fusion protein, whose molecular weight was estimated to be 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5alpha-dihydrotestosterone, and 5-androstene-3beta,17beta-diol were also catalyzed into the corresponding 17-ketosteroid at 2.4-5.9% that of estradiol oxidation. Furthermore, expressed enzyme catalyzed the reduction of estrone to estradiol, but the rate was a mere 2.3%. Human Ke 6 gene mRNA was expressed in the various tissues examined, such as brain, cerebellum, heart, lung, kidney, liver, small intestine, ovary, testis, adrenals, placenta, prostate, and stomach. Expression of human Ke 6 gene mRNA was especially abundant in prostate, placenta, and kidney. The levels in prostate and placenta were higher than that in kidney, where it is known to be expressed in large quantities.

Implications of the aquaporin-4 structure on array formation and cell adhesion.

Aquaporin-4 (AQP4) is the predominant water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder and mesial temporal lobe epilepsy. We determined the AQP4 structure by electron crystallography of double-layered, two-dimensional (2D) crystals. The structure allows us to discuss how the expression ratio between the long and short AQP4 splicing variant can determine the size of in vivo orthogonal arrays. Furthermore, AQP4 contains a short 3(10) helix in an extracellular loop, which mediates weak but specific interactions between AQP4 molecules in adjoining membranes. This finding suggests a previously unexpected role for AQP4 in cell adhesion. This notion was corroborated by expression of AQP4 in L-cells, which resulted in clustering of the cells. Our AQP4 structure thus enables us to propose models for the size regulation of orthogonal arrays and channel-mediated cell adhesion.

A new technique to co-localise membrane proteins with Homer/vesl.

The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.