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Oxalobacter formigenes is an oxalate-degrading bacterium in the gut microbiota that absorbs food-derived oxalate to use this as a carbon and energy source and thereby helps reduce the risk of kidney stone formation of the host animals 1–4. The bacterial oxalate transporter OxlT uptakes oxalate from the gut to bacterial cells and excrete formate as a degradation product, with a strict discrimination from other carboxylates that serve as nutrients 5–7. Nevertheless, the underlying mechanism remains unclear. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two different conformations, occluded and outward-facing states. The oxalate binding site contains two basic residues that form salt bridges with a dicarboxylate substrate while preventing the conformational switch to the occluded state without an acidic substrate, a ‘disallowed’ state for an antiporter 8, 9. The occluded ligand-binding pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the binding pocket are completely blocked by extensive interdomain hydrophobic and ionic interactions. Nevertheless, a molecular dynamics simulation showed that a flip of a single side chain neighbouring the substrate is sufficient to trigger the gate opening. The OxlT structure indicates the underlying metabolic interactions enabling favourable symbiosis at a molecular level.

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-β,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.

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.

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., X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.

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., 統合失調症に関わるドパミン受容体の構造解明 --副作用を抑えた薬の迅速な探索・設計が可能に--. 京都大学プレスリリース. 2020-12-24.

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.

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.

Summary 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-A 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.

Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via 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 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

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., 糖分を細胞内に輸送する膜たんぱく質の立体構造と動きを解明 -肥満やがんの抑制策に役立つ新たな知見-. 京都大学プレスリリース. 2015-10-01.

An experimental platform for serial femtosecond crystallography using an X-ray free-electron laser and its applications at SACLA are described., 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 Å was obtained.

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-A 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.

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science , this issue p. 1552

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.

Triangulating to Mechanism Cellular uptake and release of a variety of substrates are mediated by secondary transporters, but no crystal structures are known for all three fundamental states of the transport cycle, which has limited explanations for their proposed mechanisms. Shimamura et al. (p. 470 ) report a 3.8-angstrom structure of the inward-facing conformation of the bacterial sodium-benzylhydantoin transport protein, Mhp1, complementing the other two available structures. Molecular modeling for the interconversions of these structures shows a simple rigid body rotation of four helices relative to the rest of the structure in which the protein switches reversibly from outward- to inward-facing.

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H1 receptor (H1R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H1R complex with doxepin, a first-generation H1R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 4286.48, a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H1R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 1915.39 and/or Lys 179ECL2, both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H1R antagonist specificity against H1R. The common antihistamines are antagonists of histamine H1 receptor (H1R), a G-protein-coupled receptor (GPCR) that is expressed in various tissues including airway, intestinal smooth muscle and brain. The X-ray crystal structure of human H1R in the presence of doxepin, a first-generation H1R antagonist, is now reported. It shows that the drug resides in a much deeper pocket than in other aminergic GPCR structures. Analysis of drug–protein interactions should facilitate the development of antihistamines that are more selective and less likely to cause side effects than those currently available.

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite − 1. On the other hand, novel structural features were revealed in subsites + 1 and + 2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite + 1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1–3) glycosidic linkages or soluble glucan with α(1–6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.

Transporters are suggested to transport the substrates across the membrane using the alternating access mechanism in which the proteins have three functionally distinct conformational states, outward-facing, occluded and inward-facing. The structures from a secondary active transporter Mhp1 were recently determined in three states of the alternating access mechanism. Comparison of the structures shows that the conformational changes between these states are achieved by the rigid body movement of the four helices relative to the rest of the protein. The molecular basis of the alternating access mechanism is likely to be common among the LeuT superfamily.

Crystal structures of a membrane protein transporter in three different conformational states provide insights into the transport mechanism., Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-beta-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.

This paper reports the X-ray crystal structure of leukotriene C4 synthase (the pivotal enzyme for the biosynthesis of leukotriene C4), in a complex with glutathione at 3.3 Angstrom. This work may provide a structural basis for the development of new leukotriene C4 synthase inhibitors. The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis1,2, are lipid mediators of smooth muscle constriction3,4,5 and inflammation6,7, particularly implicated in bronchial asthma8,9. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein11,12 that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway14. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics17. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane α-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.

The evolutionary relationships of membrane transport proteins of the nucleobase-cation-symport (NCS-1) family from bacteria, fungi, and plants are described. The reported substrates of the NCS-1 family include nucleobases, hydantoins, and vitamins. Secondary active transport of substrate accompanied by a sodium ion, or possibly a proton, is the usual mechanism of energization. A strategy for the amplified expression, purification, and activity assays of bacterial members of the NCS-1 family is described. Conditions are given for the production of diffracting crystals of one member, the Na+-coupled transporter for aromatic hydantoins, ‘Mhp1’, from Microbacterium liquefaciens. The 3D structures of three forms of the Mhp1 protein are discussed in terms of one open-outward, one substrate-occluded, and one open-inward conformation contributing to a molecular dynamics simulation of the alternating-access model of membrane transport. The unexpected similarity of the protein fold of Mhp1 to those of transport proteins, hitherto thought to be from different evolutionary families, is discussed. 3D Structure Keywords: membrane transport protein; transporter structure; nucleobase; hydantoin

The recent crystal structure determinations of druggable class A G protein-coupled receptors (GPCRs) have opened up excellent opportunities in structure-based ligand discovery for this pharmaceutically important protein family. We have developed and validated a customized structure-based virtual fragment screening protocol against the recently determined human histamine H(1) receptor (H(1)R) crystal structure. The method combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. The optimized in silico screening approach was successfully applied to identify a chemically diverse set of novel fragment-like (≤22 heavy atoms) H(1)R ligands with an exceptionally high hit rate of 73%. Of the 26 tested fragments, 19 compounds had affinities ranging from 10 μM to 6 nM. The current study shows the potential of in silico screening against GPCR crystal structures to explore novel, fragment-like GPCR ligand space.

Volume 1 IRON Heme Proteins: Oxygen Storage and Oxygen Transport Proteins Heme Proteins: Cytochromes Heme Proteins: Cytochrome Peroxidases Heme Proteins: Cytochrome P-450 Heme Proteins: Oxidoreductases Non-Heme Proteins: Iron-Sulfur Clusters Non-Heme Proteins: Mononuclear Iron Proteins Volume 2 IRON continued Non-Heme Proteins: Dinuclear Iron Proteins Non-Heme Proteins: Iron Storage Non-Heme Proteins: Iron Transport NICKEL MANGANESE COBALT MOLYBDENUM/TUNGSTEN COPPER Cupredoxins (Type-1 Copper Proteins) Type-2 Copper Enzymes Binuclear Copper: Type-3 Copper Enzymes Binuclear Copper: CuA Copper Multicopper Enzymes Copper Storage and Transport VANADIUM.

Glucansucrases encoded byStreptococcus mutansplay essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI fromS. mutanswas overexpressed inEscherichia colistrain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space groupP21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.

Background Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Methods Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure–function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. Results As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. Conclusions The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. General significance The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.

The extended-spectrum beta-lactamases are associated with antibiotic resistance. Toho-1 R274N/R276N, a Class A beta-lactamase of CTX-M-type, efficiently hydrolyzes first generationcephalosporins (for example, cephalothin), in addition to cefotaxime, a third generation cephalosporin. However, this enzyme only marginally hydrolyzes the third generation cephalosporin ceftazidime, and the monobactam aztreonam. The deacylation defectiveness of the mutant Toho-1 E166A/R274N/R276N, which lacks the deacylation activity, results in the accumulation of the complex of an acylated-enzyme intermediate analog. For drug design, it would be useful if a quantitative prediction of a catalytic property were available without the need of enzymatic measurements. Therefore, we examined whether there is a correlation between the thermal stability of a catalytic intermediate (analog) and its kinetic parameters. First we measured the hydrolytic kinetics of the 14 species of beta-lactam antibiotics by Toho-1 R274N/R276N, and also measured the thermal stability of the accumulated acyl-intermediates of Toho-1 E166A/R274N/R276 by differential scanning calorimetry. Here we report the correlation of these parameters. The logarithm of the catalytic efficiency for Toho-1 R274N/R276N, log(k(cat)/K(m)) exhibited the best linear correlation with T(m,) which is the heat-denaturation temperature midpoint of the corresponding acylated complex of Toho-1 E166A/R274N/R276N. The correlation coefficient was 0.947, indicating that a relationship exists between the kinetic parameters and the stability of the intermediates. The results demonstrate a new method for investigating the catalytic properties of enzymes against any substrates, and a new approach to designing enzymes.

N -linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N -linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand–receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1–10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B max value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P2(1)2(1)2(1), unit-cell parameters a = 79.7, b = 101.1, c = 113.8 A). A complete data set has been collected from a single crystal to a resolution of 2.85 A with 64 741 independent observations (94% complete) and an R(merge) of 0.12. Further experimental phasing methods are under way.

Polysulfides are chains of sulfur atoms abundant in extreme environments. Some organisms reduce polysulfides, and this reaction may be coupled to respiratory processes. Now the structure of the multicomponent membrane complex that catalyzes this reaction is solved, revealing a potential proton channel that could have a role in energy conservation. Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-A resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water-filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, using polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.

Bacterial resistance to beta-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of beta-lactamases that inactivate beta-lactam antibiotics. Toho-1 is an extended-spectrum beta-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former beta-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-A resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the Omega-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn(104) and Asp(240) form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser(237) and cephalosporins probably helps to bring the beta-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.

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