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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., GLUT transporters belong to the solute carrier 2 family (SLC2) and, so far, 14 different isoforms (GLUT1-GLUT14) have been identified (1,2). Except for GLUT13, GLUT transporters are uniporters, which facilitate [...]

The crystal structure of the bacterial protein YidC is reported, together with a structure-based functional analysis, providing insight into the role of YidC in inserting single-spanning membrane proteins into the membrane. Structure of YidC from Bacillus halodurans The bacterial protein YidC, a homologue of mitochondrial Oxa1 and chloroplast Alb3, is not just a chaperone that facilitates proper folding and membrane topology of its membrane-protein substrates, in cooperation with the Sec machinery. It also inserts several single- or double-spanning membrane proteins into the membrane independently of Sec. Osamu Nureki and colleagues present the long-awaited structure of YidC, which provides insights into the second role of this membrane protein. The structure suggests that YidC does not adopt a polypeptide-conducting channel-like architecture. Instead, a novel fold within the protein forms a positively charged hydrophilic groove. The authors, using structure-based functional analysis, reveal that electrostatic interactions between a conserved Arg residue in the groove and the acidic residues at the N-terminal region of a substrate protein are essential for the YidC-mediated insertion of the substrate into the membrane. Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively.sup.1,2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG.sup.3,4,5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane., Author(s): Kaoru Kumazaki [sup.1] [sup.2] , Shinobu Chiba [sup.3] , Mizuki Takemoto [sup.1] [sup.2] , Arata Furukawa [sup.4] , Ken-ichi Nishiyama [sup.5] , Yasunori Sugano [sup.4] , Takaharu Mori [sup.6] [...]

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states (1-13). Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The [A.sub.2A] adenosine receptor ([A.sub.2A]AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain (14). Here we report the raising of a mouse monoclonal antibody against human [A.sub.2A]AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of [A.sub.2A]AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of [A.sub.2A]AR 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 (1) and to CDR-3 of the nanobody in the active [β.sub.2]-adrenergic receptor structure (2), but locks [A.sub.2A]AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors., The structures of G-protein-coupled receptors (GPCRs) in an inactive conformation solved recently (3,12) greatly advance our understanding of the molecular signalling mechanisms of the receptors. The first details of GPCR [...]

Author(s): Reiya Taniguchi [1, 2]; Asuka Inoue [3, 4]; Misa Sayama [5]; Akiharu Uwamizu [3]; Keitaro Yamashita [6]; Kunio Hirata [4, 6]; Masahito Yoshida [7]; Yoshiki Tanaka [8]; Hideaki E. [...]

Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement. [ABSTRACT FROM AUTHOR]

Abstract: The generation of antibodies against self-antigens or antigens having a high degree of structural homology with self-antigens is a difficult task because of immunological tolerance. CD4+CD25+ regulatory T cells play an important role in maintaining peripheral tolerance. Sakaguchi et al. previously reported that the transfusion of CD25+ cell-depleted mouse splenocytes into syngeneic nude mice results in a breaking of peripheral tolerance that leads to the development of autoimmunity. In this study, we attempted to apply this mouse model to the generation of antibodies against self-antigens. We depleted CD25+ cells from BALB/c mouse splenocytes and transferred the rest of the cells into syngeneic nude mice. The animals were immunized with mouse thyroglobulin. We observed a significant increase of the anti-mouse thyroglobulin antibody titer in the group of mice immunized twice within 10days after the cell transfer (P <0.05). From these mice, we established hybridoma cell lines producing anti-mouse thyroglobulin monoclonal antibodies, including a clone with a dissociation constant of 10−8 M. Control nude mice which received CD25+ cell-containing BALB/c splenocytes did not produce anti-mouse thyroglobulin antibodies. When the CD25–cell-transferred nude mice were immunized with mouse Gα12, another self-antigen, anti-Gα12 antibodies were produced in the sera. This method should prove highly useful in the generation of antibodies against self-antigens or antigens for which the structure is highly conserved across species. [Copyright &y& Elsevier]

Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H+ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters. The multidrug resistance transporter mediated efflux of antibiotics from the bacterial cytoplasm represents a major challenge to medicine. Here authors solve the X-ray crystallographic structure of the drug/H+ antiporter MdfA from Escherichia coli and shed light on the conformational switching mechanism. [ABSTRACT FROM AUTHOR]

A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties.

YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P21, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.