Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota.

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

S1PR3-G12-biased agonist ALESIA targets cancer metabolism and promotes glucose starvation.

Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G12-biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy.

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone.

Angiotensin II (AngII) is a peptide hormone that plays a key role in regulating blood pressure, and its interactions with the G protein-coupled receptors, AngII type-1 receptor (AT1R) and AngII type-2 receptor (AT2R), are central to its mechanism of action. We solved the crystal structure of human AT2R bound to AngII and its specific antibody at 3.2-Å resolution. AngII (full agonist) and [Sar1, Ile8]-AngII (partial agonist) interact with AT2R in a similar fashion, except at the bottom of the AT2R ligand-binding pocket. In particular, the residues including Met1283.36, which constitute the deep end of the cavity, play important roles in angiotensin receptor (ATR) activation upon AngII binding. These differences that occur upon endogenous ligand binding may contribute to a structural change in AT2R, leading to normalization of the non-canonical coordination of helix 8. Our results will inform the design of more effective ligands for ATRs.

High-viscosity sample-injection device for serial femtosecond crystallography at atmospheric pressure.

A sample-injection device has been developed at SPring-8 Angstrom Compact Free-Electron Laser (SACLA) for serial femtosecond crystallography (SFX) at atmospheric pressure. Microcrystals embedded in a highly viscous carrier are stably delivered from a capillary nozzle with the aid of a coaxial gas flow and a suction device. The cartridge-type sample reservoir is easily replaceable and facilitates sample reloading or exchange. The reservoir is positioned in a cooling jacket with a temperature-regulated water flow, which is useful to prevent drastic changes in the sample temperature during data collection. This work demonstrates that the injector successfully worked in SFX of the human A2A adenosine receptor complexed with an antagonist, ZM241385, in lipidic cubic phase and for hen egg-white lysozyme microcrystals in a grease carrier. The injection device has also been applied to many kinds of proteins, not only for static structural analyses but also for dynamics studies using pump-probe techniques.

Structural basis for the substrate recognition of aminoglycoside 7''-phosphotransferase-Ia from Streptomyces hygroscopicus.

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7''-phosphotransferase-Ia [APH(7'')-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7'' and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7'')-Ia and APH(4)-Ia, the crystal structure of APH(7'')-Ia complexed with HygB is reported. The overall structure of APH(7'')-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7'')-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7'')-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.

Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine.

Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs.

Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog.

Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the 'core' binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the 'extended' binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.

Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA.

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.

A three-dimensional movie of structural changes in bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent.

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1.

Pembrolizumab is an FDA-approved therapeutic antibody that targets the programmed cell death-1 (PD-1) to block the immune checkpoint pathway for the treatment of various types of cancer. It receives remarkable attention due to the high degree of efficacy. Very recently, the crystal structure of the Fab fragment of pembrolizumab (PemFab) in complex with the extracellular domain of human PD-1 (PD-1ECD) was reported at a resolution of 2.9 Å. However, this relatively low-resolution structural data fails to provide sufficient information on interfacial water molecules at the binding interface that substantially contribute to affinity and specificity between the therapeutic antibody and target. Here, we present the independently determined crystal structure of the Fv fragment of pembrolizumab (PemFv) in complex with the PD-1ECD at a resolution of 2.15 Å. This high-resolution structure allows the accurate mapping of the interaction including water-mediated hydrogen bonds and provides, for the first time, a coherent explanation of PD-1 antagonism by pembrolizumab. Our structural data also provides new insights into the rational design of improved anti-PD-1 therapeutics.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance.

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Šis successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

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.

Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser.

An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Šwas obtained.

Grease matrix as a versatile carrier of proteins for serial crystallography.

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.

The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.