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Purple phototrophic bacteria are ancient anoxygenic phototrophs and attractive research tools because they capture light energy in the near-infrared (NIR) region of the spectrum and transform it into chemical energy by way of uphill energy transfers. The heart of this reaction occurs in light-harvesting 1-reaction center (LH1-RC) complexes, which are the simplest model systems for understanding basic photosynthetic reactions within type-II (quinone-utilizing) reaction centers. In this Perspective, we highlight structure-function relationships concerning unresolved fundamental processes in purple bacterial photosynthesis, including the diversified light-harvesting capacity of LH1-associated BChl molecules, energies necessary for photoelectric conversion in the RC special pairs, and quinone transport mechanisms. Based on recent progress in the spectroscopic and structural analysis of LH1-RC complexes from a variety of purple phototrophs, we discuss several key factors for understanding how purple bacteria resource light energy in the inherently energy-poor NIR region of the electromagnetic spectrum.

We have previously reported the crystal structures of endothelin-1 (ET-1)-bound, ligand-free, and antagonist bosentan-bound forms of the thermostabilized ET type B receptor (ETB). Although other agonist-bound structures of ETB have been determined, the interactions for high-affinity binding and ETB receptor activation, as well as the roles of rearrangement of the hydrogen-bond network surrounding the ligand in G protein activation, remain elusive. ET-1, a 21-amino acid residue peptide, plays fundamental roles in basal vascular tone, sodium balance, cell proliferation, and stress-responsive regulation. We studied the interactions between the ET-1(8–21) peptide and ETB in the ligand binding and activation of ETB using a series of Ala-substituted ET-1(8–21) analogues and the mutated ETB. We found that while D8, L17, D18, I20, and W21 were responsible for high-affinity binding and potent G protein activation, Y13 and F14 in the helical region of ET-1 are prerequisites for the full activation of ETB via interactions near the extracellular side. Furthermore, we introduced the mutation into the residues around the ET-1 binding pocket of ETB. The results showed that while S1843.35, W3366.48, N3787.45, and S3797.46 in a conserved polar network are required for full activation, N1191.50, D1472.50, and N3827.49 are essential for G protein activation via direct interactions after rearrangement upon ET-1 binding. These results demonstrate that both interactions near the extracellular side and within the transmembrane helices with ET-1 play crucial roles in the full activation of the ETB receptor.

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Commun. 12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 Å resolution and from an LH1-RC monomer lacking protein-U (ΔU) at 2.64 Å resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The ΔU LH1-RC monomer revealed a half-moon-shaped structure containing 11 α- and 10 β-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of αβ-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.

We present a cryo-EM structure of the monomeric light-harvesting-reaction center (LH1-RC) core complex from photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its pores. Our findings offer a new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

Rhodospirillum (Rsp.) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl aG) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 A resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αβ-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the QA site in the RC. The geranylgeraniol side chains of BChl aG coordinated by LH1 β-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 α-associated BChls aG. The structure also revealed key protein-protein interactions in both N- and C-terminal regions of the LH1 αβ-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex.

In Saccharomyces cerevisiae, the glyoxylate cycle is controlled through the posttranslational regulation of its component enzymes, such as isocitrate lyase (ICL), which catalyzes the first unique step of the cycle. The ICL of S. cerevisiae (ScIcl1) is tagged for proteasomal degradation through ubiquitination by a multisubunit ubiquitin ligase (the glucose-induced degradation-deficient (GID) complex), whereas that of the pathogenic yeast Candida albicans (CaIcl1) escapes this process. However, the reason for the ubiquitin targeting specificity of the GID complex for ScIcl1 and not for CaIcl1 is unclear. To gain some insight into this, in this study, the crystal structures of apo ScIcl1 and CaIcl1 in complex with formate and the cryogenic electron microscopy structure of apo CaIcl1 were determined at a resolution of 2.3, 2.7, and 2.6 A, respectively. A comparison of the various structures suggests that the orientation of N-terminal helix α1 in S. cerevisiae is likely key to repositioning of ubiquitination sites and contributes to the distinction found in C. albicans ubiquitin evasion mechanism. This finding gives us a better understanding of the molecular mechanism of ubiquitin-dependent ScIcl1 degradation and could serve as a theoretical basis for the research and development of anti-C. albicans drugs based on the concept of CaIcl1 ubiquitination.

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has already been applied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αβ-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αβ-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and β-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside., Here the authors report a cryo-EM structure of the light-harvesting-reaction center complex (LH1- RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970, providing insights into the mechanisms that underlie the absorbance properties of both the LH1 and the RC of this spectrally unusual purple bacterium.

Cryo-EM structures of the undocked INX-6 hemichannels reveal a pore obstruction and N-terminal rearrangement in lipids., Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo–electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

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 (β-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.

Rhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αβ-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general., Rhodobacter (Rba.) sphaeroides is a model organism for studying bacterial photosynthesis. Here, the authors present the 2.9 Å cryo-EM structure of the monomeric light-harvesting-reaction center core complex from Rba. sphaeroides strain IL106, which revealed the position and conformation of PufX and the presence of an additional component protein-U, an integral membrane protein.

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-A and 2.2-A resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

Claudins are four-transmembrane proteins that constitute the backbone of tight junction strands via self-polymerization in the apicolateral membranes of epithelial cells. Together with their cell-cell adhesion function, claudin proteins form the paracellular barrier and/or channels through epithelial cell sheets whose permeability is primarily dependent on the claudin subtype. Recently determined crystal structures of several claudins revealed the unique claudin fold of four transmembrane helices in a left-handed helical bundle with an extracellular β-sheet domain. Here, we focus on the structural basis of the intermolecular interactions between claudin molecules and between the Clostridium perfringens enterotoxin and its receptor claudins.

The vasoconstricitor peptide endothelin and its G-protein-coupled receptor (GPCR) are involved in the development of various diseases, such as pulmonary arterial hypertension, so are important therapeutic targets. In this manuscript, the authors report the X-ray crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism of the isopeptide selectivity between endothelin-1 and endothelin-3. Comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A GPCRs.

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD

How a toxin makes epithelial sheets leaky The entire human body and its many compartments are shielded from their external environments by the barrier function of epithelial cell sheets. The paracellular barrier function of tight junctions (TJs) is critical for maintaining homeostasis in any multicellular organism, especially in the skin and internal organs and at the blood-brain barrier. One of the major components of TJs is a family of adhesive membrane proteins known as claudins. Several members of the claudin family are receptors for the bacterial toxin Clostridium perfringens enterotoxin. This toxin often causes food-borne illness both in humans and animals. Saitoh et al. crystallized a complex between the toxin and a claudin that reveals just how the toxin damages epithelial barriers (see the Perspective by Artursson and Knight). Science , this issue p. 775 ; see also p. 716

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ET

Background: The gastric H+,K+-ATPase proton pump achieves gastric acid secretion. Results: A newly determined (SCH)E2·MgF structure represents a hybrid conformation of (SCH)E2·BeF and luminal-closed (Rb+)E2·AlF states. Conclusion: Comparison of E2P-related structures revealed that the A-M2 linker importantly contributes to conformational changes of the enzyme. Significance: A-M2 linker-mediated conformational change is conserved between H+,K+-ATPase and Ca2+-ATPase and perhaps other P-type ATPases., Gastric H+,K+-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K+-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H+,K+-ATPase is consistent with that observed in sarcoplasmic reticulum Ca2+-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.

Bovine heart NADH:ubiquinone oxidoreductase (complex I), which is the largest (about 1 MDa) membrane protein complex in the mitochondrial respiratory chain, catalyzes the electron transfer from NADH to ubiquinone, coupled with proton pumping. We have crystallized bovine complex I in reconstituted lipid bilayers and obtained a three-dimensional density map by the electron crystallographic analysis of a single negatively stained two-dimensional crystal. The asymmetric unit with dimensions of a = 388 Å, b = 129 Å and γ = 90° contains two molecules and is of P1 symmetry. Structural differences between the two molecules indicate flexibility of the hydrophilic domain relative to the membrane-embedded domain.

Received 7 June 2013; accepted 21 June 2013Abstract Electron crystallography is an important method for determining the structure of membrane proteins. In this paper, we show the impact of a carbon sandwich preparation on the preservation of crystalline sample quality, using characteristic examples of two-dimensional (2D) crystals from gastric H+,K+-ATPase and their analyzed images. Compared with the ordinary single carbon support film preparation, the carbon sandwich preparation dramatically enhanced the resolution of images from flat sheet 2D crystals. As water evaporation is restricted in the carbon-sandwiched specimen, the improvement could be due to the strong protective effect of the retained water against drastic changes in the environment surrounding the specimen, such as dehydration and increased salt concentrations. This protective effect by the carbon sandwich technique helped to maintain the inherent and therefore best crystal conditions for analysis. Together with its strong compensation effect for the image shift due to beam-induced specimen charging, the carbon sandwich technique is a powerful method for preserving crystals of membrane proteins with larger hydrophilic regions, such as H+,K+-ATPase, and thus constitutes an efficient and high-quality method for collecting data for the structural analysis of these types of membrane proteins by electron crystallography.

Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families., Gap junctions have critical roles in maintaining homeostasis in multicellular organisms. Here the authors present cryo-EM structures of the C. elegans innexin-6 gap junction channel, revealing high structural similarity to human connexin 26 despite a different oligomeric number and lack of sequence similarity.

Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue- and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na(+), similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms.

The peptide hormone endothelin, produced by the vascular endothelium, is involved in several physiological functions, including maintenance of vascular tone and humoral homeostasis. Endothelin transmits signals through the endothelin receptor, a G-protein-coupled receptor. Structural studies of the endothelin type B receptor (ETBR) have been unsuccessful due to its structural flexibility and instability in detergent-solubilized solution. To overcome these problems, we explored thermostabilization of human ETBR by establishing an ETBR expression system in Escherichia coli, followed by systematic alanine scanning mutagenesis. Among 297 point mutations, 11 thermostabilizing residues were selected and further mutated to other amino acids. The thermostability indices of these residues, represented by the ratios of endothelin-1 (ET-1) binding activities with or without heat treatment at 27°C for 30min in a ligand-free form, were compared. The ligand affinity and apparent melting temperature (Tm) of the five most thermostable mutants, R124Y, D154A, K270A, S342A, and I381A, were then examined. The apparent Tm of three single mutants, R124Y, D154A, and K270A, was approximately 7°C higher than that of the wild type. The apparent Tm value of a combination of the five residues, named the Y5 ETBR mutant, was 17°C higher than that of the wild type. The Y5 ETBR mutant exhibited an affinity for ET-1 and activated Gq similar to the wild type. Further investigation of the pharmacological properties affected by combinatorial mutations of ET-1, ET-3, TxET-1, and K8794 suggested that Y5 ETBR is highly suitable for representing a ligand-free form of ETBR and is potentially applicable for studying an ET-1-bound form.

Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.

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.

Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. In order to characterize the phosphorylation events on H(+),K(+)-ATPase, the properties of fluorinated phosphate analogs [XFs, e.g. aluminum fluoride (AlF), beryllium fluoride (BeF) and magnesium fluoride (MgF)], and the structural differences induced by XFs were investigated. The addition of divalent cations to the XF-inhibited H(+),K(+)-ATPase restores the activity of the AlF- or MgF-inhibited, but not of the BeF-inhibited enzyme, although limited trypsin digestion reveals that they assume the same E(2)P-like state. To clarify the conformational differences induced by XFs, the structure of BeF-bound H(+),K(+)-ATPase was analyzed at 8A resolution. The structure is almost identical to the previously reported AlF-bound E(2)P structure, unlike the distinctive X-ray structure of BeF-bound SERCA, in which the luminal gate was observed to be widely opened. Since the analyzed structure of the H(+),K(+)-ATPase revealed that both AlF and BeF-bound to the P domain were not exposed to the solvent, the dissociation of XFs induced by divalent cations could be interpreted in terms of stability against thermal fluctuations. Furthermore, the conformational differences found between the cytoplasmic domains of H(+),K(+)-ATPase and SERCA provide a framework to understand the characteristic mechanism, by which divalent cations reactivate the XF-inhibited H(+),K(+)-ATPase.

Acid secretion by the stomach results in a pH of about 1. This highly acidic environment is essential for digestion and also acts as a first barrier against bacterial and viral infections. Conversely, too much acid secretion causes gastric ulcer. The mechanism by which this massive proton gradient is generated is of considerable biomedical interest. In this review, we introduce the first molecular model for this remarkable biological phenomenon. The structure of H(+),K(+)-ATPase at 6.5 A resolution was determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the N-terminal tail of the beta-subunit functions as a "ratchet", preventing inefficient transport and reverse-flow of protons. We can thus provide a mechanistic explanation for how the H(+),K(+)-ATPase can generate a million-fold proton gradient across the gastric parietal cell membrane, the highest cation gradient known in any mammalian tissue.

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.

The gastric H+,K+-ATPase is an ATP-driven proton pump responsible for generating a million-fold proton gradient across the gastric membrane. We present the structure of gastric H+,K+-ATPase at 6.5 Å resolution as determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic α-subunit and the non-catalytic β-subunit in a pseudo-E2P conformation. Different from Na+,K+-ATPase, the N-terminal tail of the β-subunit is in direct contact with the phosphorylation domain of the α-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the β-subunit N-terminus prevents the reverse reaction from E2P to E1P, which is likely to be relevant for the generation of a large H+ gradient in vivo situation.

The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1PADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E1 to the E1P x ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E1 and E1P x ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E1 and E1P x ADP states present in the 2D crystals,showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase.

Gated gap junction channels are important cellular conduits for establishing and maintaining intercellular communication. The three-dimensional structure of a mutant human connexin 26 (Cx26M34A) by electron cryocrystallography revealed a plug-like density in the channel pore suggesting that physical blockage of the pore may be one mechanism of closure (Oshima et al. 2007, Proc Natl Acad Sci USA 104: 10034-10039). However, it remains to be determined what part of the sequence contributes to the plug. Here, we present the projection structure of an N-terminus deletion of Cx26M34A missing amino acids 2 to 7 (Cx26M34Adel2-7) crystallized in the same two-dimensional crystal form. A 10 A resolution projection map of Cx26M34Adel2-7 revealed that the plug density was dramatically reduced in comparison with that found in full-length Cx26 channel. The difference map between the deletion and full-length Cx26M34A channels strongly suggests that the N-terminus of connexin contributes to the plug for the physical closure of gap junction channels.