Tomoya Hino

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AAR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A2AAR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β2-adrenergic receptor structure, but locks A2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase

Dissecting Nitric Oxide Reductase Bacterial breakdown of nitrogen compounds in soil and the oceans provides the largest emission source of the greenhouse gas, nitrous oxide (N2O). A key enzyme in this process is nitric oxide reductase (NOR), which catalyzes the reduction of nitric oxide (NO) to N2O. Hino et al. (p. 1666, published online 25 November; see the Perspective by Moënne-Loccoz and Fee) now describe the crystal structure of NOR from Pseudomonas aeruginosa. Consistent with their evolutionary relatedness, the transmembrane region topology and arrangement of metal centers in NOR are similar to those in cytochrome oxidases, key enzymes in aerobic respiration. A structural comparison gives insight into the features that allow conversion between nitric oxide and oxygen reduction. Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N2O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (FeB) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.

Structure and mechanism of the mammalian fructose transporter GLUT5

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a ‘gated-pore’ transport mechanism in such monosaccharide transporters.

NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa CHARACTERIZATION OF REACTION INTERMEDIATES THAT APPEAR IN THE SINGLE TURNOVER CYCLE

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N2O at the binuclear catalytic center consisting of high spin heme b3 and non-heme FeB. The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous FeB–NO and the penta-coordinated ferrous heme b3–NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N2O formation, in good agreement with the so-called “trans” mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the μ-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.

Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus

The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N2O, has been characterized at 2.5 Å resolution. The overall fold of qNOR is similar to that of cytochrome c–dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

Crystal structure of the anion exchanger domain of human erythrocyte band 3.

Getting rid of carbon dioxide In mammals, red blood cells deliver oxygen to tissues and remove carbon dioxide. Key to this essential process is a membrane protein called anion exchanger 1 (AE1) which transports bicarbonate (formed from carbon dioxide) out of red blood cells in exchange for chloride. This decreases the pH inside the blood cells, so that oxygen is released from hemoglobin and can diffuse into tissues. Arakawa et al. report the crystal structure of the transmembrane anion exchanger domain of AE1, which includes 14 transmembrane helices. The structure provides a basis for understanding the effects of mutations that lead to red blood cell diseases and also gives insight into the mechanism of ion transport. Science, this issue p. 680 The structure of a key red blood cell membrane protein provides a basis for understanding mutations that lead to red blood cell diseases. Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease.

Molecular structure and function of bacterial nitric oxide reductase

The crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b(3) and non-heme Fe(B)). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca(2+) sandwiched between two heme propionates from heme b and b(3), and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.

Generation of functional antibodies for mammalian membrane protein crystallography.

Membrane proteins act as gateways to cells, and they are responsible for much of the communication between cells and their environments. Crystallography of membrane proteins is often limited by the difficulty of crystallization in detergent micelles. Co-crystallization with antibody fragments has been reported as a method to facilitate the crystallization of membrane proteins; however, it is widely known that the generation of mouse monoclonal antibodies that recognize the conformational epitopes of mammalian integral membrane proteins is typically difficult. Here, we present our protocols to generate functional mouse antibodies for the membrane protein crystallography, which have enabled us to solve crystal structures of mammalian receptors and transporters complexed with antibody fragments.

Structural basis for nitrous oxide generation by bacterial nitric oxide reductases.

The crystal structure of the bacterial nitric oxide reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.

Molecular dynamics simulations reveal proton transfer pathways in cytochrome C-dependent nitric oxide reductase.

Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666–70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb 3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.