Search

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.

G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H 1 receptor (H 1 R) expressed in Pichia pastoris . Here, we report the expression and purification procedures of recombinant H 1 R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.

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.

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.

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.

In the crystal structure of the native GroEL·GroES·substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL·GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K)·substrate complex failed to bind GroES to produce a stable GroEL(L309K)·GroES·substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K)·GroES·substrate complex was stabilized with BeFx, but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu309), appears to play a critical role in encapsulation of the substrate.

Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl− and HCO 3 - across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.9% identity and 40.3% similarity. PcAEP was overexpressed by introducing the PcAEP gene into the genome of Pichia pastoris. As a result, PcAEP localized in the membrane of P. pastoris and was solubilized successfully by n-dodecyl-β- d -maltoside. His-tagged PcAEP was purified as a single band on SDS–PAGE using immobilized metal affinity chromatography and gel filtration chromatography. Purified PcAEP was found to bind to SITS, an inhibitor of the AE family, suggesting that the purified protein is folded properly. PcAEP expressed and purified using the present system could be useful for biological and structural studies of the anion exchange family of proteins.

Background Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. Results The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. Conclusion Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

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.

Purpose of work Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.

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.

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.

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

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.

A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 A resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed alpha-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I.

The chaperonins GroEL and GroES are essential mediators of protein folding. GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity). We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution. Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals. The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex. The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry. As a result, the GroEL-GroES interface differs considerably from the previously reported E. coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES.

The chaperonin GroEL-GroES (GroEL/ES) complex from a thermophilic eubacteria, Thermus thermophilus HB8, has been purified and crystallized. The GroEL/ES complex is known to be composed of 14 identical GroEL subunits (58 kDa) and seven identical GroES subunits (11 kDa). The GroEL/ES complex crystals belong to the triclinic space group P1, with unit-cell parameters a = 140.4, b = 156.4, c = 273.1 A, alpha = 82.9, beta = 85.4, gamma = 68.5 degrees. The crystal asymmetric unit contains two molecules (MW = 885 kDa). One data set to 3.0 A resolution, with 383 652 independent observations (89.3% complete) and an R(merge) of 0.08, has been collected from a single crystal. A molecular-replacement solution was obtained using the structure of the GroEL/ES complex from Escherichia coli as a search model.

Background Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. Results We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. Conclusions We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.