The DROL1 subunit of U5 snRNP in the spliceosome is specifically required to splice AT-AC-type introns in Arabidopsis.

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA sequencing analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+ /H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini and that this plays an important role in plant growth and development.

Cell-based flow cytometry assay for simultaneous detection of multiple autoantibodies in a single serum sample.

Accurate serologic evaluation of autoantibodies in patients with autoimmune diseases is critical. In the present study, we established a live cell-based assay for simultaneous detection of multiple autoantibodies in a single serum sample. Autoantibody seropositivity was determined by 3-color flow cytometry using live Chinese hamster ovary cells transiently expressing a target protein of interest fused to enhanced green fluorescent protein and labeled with Alexa Fluor 647 and Hoechst 33342. As a representative example, we applied the strategy for simultaneous detection of 2 recently established biomarkers for central nervous system autoimmune inflammatory demyelinating disorders, anti-aquaporin-4 autoantibody and anti-myelin oligodendrocyte glycoprotein autoantibody, in a single serum sample. This analysis revealed the coexistence of these 2 autoantibodies. We demonstrated that this assay can simultaneously detect 3 different autoantibodies. We propose a quadrant gating strategy of flow cytometry contour plots to clearly distinguish seropositive sera from seronegative sera regardless of the extent of the background signal level or the autoantibody titer. This novel and practical method using a combination of fluorescent proteins and fluorochromes to simultaneously detect multiple autoantibodies improves the efficiency of evaluating serum samples, and therefore provides significant benefits to both the patient and the healthcare professionals performing autoantibody testing.

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.

Structural insight into the activation mechanism of MrgD with heterotrimeric Gi-protein revealed by cryo-EM.

MrgD, a member of the Mas-related G protein-coupled receptor (MRGPR) family, has high basal activity for Gi activation. It recognizes endogenous ligands, such as ß-alanine, and is involved in pain and itch signaling. The lack of a high-resolution structure for MrgD hinders our understanding of whether its activation is ligand-dependent or constitutive. Here, we report two cryo-EM structures of the MrgD-Gi complex in the ß-alanine-bound and apo states at 3.1 Å and 2.8 Å resolution, respectively. These structures show that ß-alanine is bound to a shallow pocket at the extracellular domains. The extracellular half of the sixth transmembrane helix undergoes a significant movement and is tightly packed into the third transmembrane helix through hydrophobic residues, creating the active form. Our structures demonstrate a structural basis for the characteristic ligand recognition of MrgD. These findings provide a framework to guide drug designs targeting the MrgD receptor.

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.

Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.

Reconstruction of the P2X(2) receptor reveals a vase-shaped structure with lateral tunnels above the membrane.

In response to the intercellular messenger ATP, P2X receptors transfer various sensory information, including pain. Here we have reconstructed the structure of the P2X(2) receptor at 15 A resolution from more than 90,000 particle images, taken with a cryo-electron microscope equipped with a helium-cooled stage. This three-dimensional depiction, presumably in a closed state, revealed an elongated vase-shaped structure 202 A in height and 160 A in major diameter. The extracellular and transmembrane domains present a two-layered structure, in which a sparse outer layer surrounds a pore-forming inner density. The decreased diameter of a putative ion-conducting pathway at the middle of the membrane was considered to be the narrowest part of the pore, which has been predicted from electrophysiological studies. The sparse, extended structure of the P2X(2) receptor indicates a loose assembly of subunits, which could be a basis for the activation-dependent pore dilation of P2X receptors.

Unusual thermal disassembly of the SPFH domain oligomer from Pyrococcus horikoshii.

Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping.

Acetazolamide reversibly inhibits water conduction by aquaporin-4.

Aquaporin-4 (AQP4) has been implicated in cytotoxic brain edema resulting from water intoxication, brain ischemia or meningitis. AQP4 inhibitors suitable for clinical use would thus be expected to help protect against brain edema. Here, we report the effect of inhibitors on water conduction by AQP4 and AQP1 reconstituted into liposomes. Acetazolamide (AZA), an inhibitor of sulfonamide carbonic anhydrase (CA), reversibly inhibits water permeation through AQP4, but not through AQP1. Methazolamide (MZA), another sulfonamide CA inhibitor similar in chemical structure to AZA, shows no significant effect on water conduction by AQP4 or AQP1. Our results thus demonstrate that AZA acts as a reversible inhibitor for AQP4-mediated water conduction and indicate that AZA is specific, at least to some degree, for AQP4. AZA may thus serve as a lead compound for the development of AQP4-specific inhibitors for clinical applications.

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.

Aquaporin-11 containing a divergent NPA motif has normal water channel activity.

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.

Sendai virus F glycoprotein induces IL-6 production in dendritic cells in a fusion-independent manner.

We previously reported that inactivated Sendai virus particle (hemagglutinating virus of Japan envelope; HVJ-E) has anti-tumor effects by eliciting IL-6 production in dendritic cells (DCs). In the present study, we investigated which components of HVJ-E elicit IL-6 production. HVJ-E containing F0 protein inactive for virus envelope-cell membrane fusion enhanced IL-6 production. Reconstituted liposomes containing F protein stimulated IL-6 production. The antibody against F protein inhibited IL-6 secretion by HVJ-E. When carbohydrate chains of the F glycoprotein were removed, HVJ-E lost the ability to stimulate IL-6 secretion. These results suggest that F glycoprotein is required for IL-6 production in DCs.

The TRPC3 channel has a large internal chamber surrounded by signal sensing antennas.

Transient receptor potential (TRP) channels are intrinsic sensors adapted for response to all manner of stimuli both from inside and from outside the cell. Within the TRP superfamily, the canonical TRP-3 (TRPC3) has been widely studied and is involved in various biological processes such as neuronal differentiation, blood vessel constriction, and immune cell maturation. Upon stimulation of surface membrane receptors linked to phospholipase C, TRPC3 mediates transmembrane Ca(2+) influx from outside the cell to control Ca(2+) signaling, in concert with the Ca(2+) release from internal stores. The structural basis of TRP superfamily has, however, been poorly understood. Here we present a structure of the TRPC3 at 15 A resolution. This first 3D depiction of TRP superfamily was reconstructed from 135,909 particle images obtained with cryo-electron microscopy. The large intracellular domain represents a "nested-box" structure: a wireframe outer shell is functionable as sensors for activators and modulators, and a globular inner chamber may modulate ion flow, since it is aligned tandem along the central axis with the dense membrane-spanning core. The transmembrane domain demonstrates a pore-forming property. This structure implies that the TRP superfamily has diversely evolved as sensors specialized for various signals, rather than as simple ion-conducting apparatuses.

[Aquaporin-4].

In human body, there are thirteen water channels but their expression patterns are tissue specific. Aquaporin-4 (AQP4) is a predominantly expressed water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder, and mesial temporal lobe epilepsy. Recently it was reported that IgG of optic-spinal multiple sclerosis patients bound to AQP4. In order to reveal the function of AQP4, we determined the atomic structure of AQP4 by electron crystallography of double layered two-dimensional crystals. In double layered crystal, each single layered crystal contacts by a short 310 helix in the extracellular loop C. It would suggest that AQP4 shows the weak adhesive activity between adjoining membranes. This is correlated to immunogold labeling of AQP4 in glial lamellae localizing the protein areas where the membranes are separated but also all along junctional regions. Furthermore, from the freeze fracture replica labeling and the mutational experiment, the palmitoylation of N-terminal cysteine residues makes orthogonal array structure unstable on Chinese hamster Ovary (CHO) cell membrane. These findings suggest that there must be the complicated mechanism for control of water content relevant to AQP4 within the brain.

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.

Two-dimensional crystal structure of aquaporin-4 bound to the inhibitor acetazolamide.

Acetazolamide (AZA) reduces the water permeability of aquaporin-4, the predominant water channel in the brain. We determined the structure of aquaporin-4 in the presence of AZA using electron crystallography. Most of the features of the 5-Å density map were consistent with those of the previously determined atomic model. The map showed a protruding density from near the extracellular pore entrance, which most likely represents the bound AZA. Molecular docking simulations supported the location of the protrusion as the likely AZA-binding site. These findings suggest that AZA reduces water conduction by obstructing the pathway at the extracellular entrance without inducing a large conformational change in the protein.

Crystal structure of the Homer 1 family conserved region reveals the interaction between the EVH1 domain and own proline-rich motif.

PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction. Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD. It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family. The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal. The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1. The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins.

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains.

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.

Two alternative conformations of a voltage-gated sodium channel.

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.

Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration.