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Audray Harris,
Mario J Borgnia, Dan Shi,
Alberto Bartesaghi,
Haifeng He,
Robert Pejchal,
Yun Kenneth Kang,
Rafael Depetris,
Andre J Marozsan,
Rogier W Sanders,
Per Johan Klasse,
Jacqueline L S Milne,
Ian A Wilson,
William C Olson,
John P Moore,
Sriram Subramaniam
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ABSTRACT: The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.
Proceedings of the National Academy of Sciences 06/2011; 108(28):11440-5. · 9.68 Impact Factor
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Richard L Felts,
Kedar Narayan,
Jacob D Estes, Dan Shi,
Charles M Trubey,
Jing Fu,
Lisa M Hartnell,
Gordon T Ruthel,
Douglas K Schneider,
Kunio Nagashima,
Julian W Bess,
Sina Bavari,
Bradley C Lowekamp,
Donald Bliss,
Jeffrey D Lifson,
Sriram Subramaniam
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ABSTRACT: The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.
Proceedings of the National Academy of Sciences 07/2010; 107(30):13336-41. · 9.68 Impact Factor
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Richard L. Felts,
Kedar Narayan,
Jacob D. Estes, Dan Shi,
Charles M. Trubey,
Jing Fu,
Lisa M. Hartnell,
Gordon T. Ruthel,
Douglas K. Schneider,
Kunio Nagashima,
Julian W. Bess Jr,
Sina Bavari,
Bradley C. Lowekamp,
Donald Bliss,
Jeffrey D. Lifson,
Sriram Subramaniam
[show abstract]
[hide abstract]
ABSTRACT: The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known
as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed
the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature
dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived
from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial
extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic
cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively
at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells
from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer
by T cells highlight unique aspects of cell-cell HIV transmission.
Proceedings of the National Academy of Sciences 07/2010; 107(30):13336-13341. · 9.68 Impact Factor
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Adam E Bennett,
Kedar Narayan, Dan Shi,
Lisa M Hartnell,
Karine Gousset,
Haifeng He,
Bradley C Lowekamp,
Terry S Yoo,
Donald Bliss,
Eric O Freed,
Sriram Subramaniam
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ABSTRACT: HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of approximately 150-200 nm, and lengths of up to approximately 5 microm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.
PLoS Pathogens 09/2009; 5(9):e1000591. · 9.13 Impact Factor
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ABSTRACT: Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at approximately 30nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the "hard" (siliceous) and "soft" (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system.
Journal of Structural Biology 04/2009; 166(3):316-28. · 3.41 Impact Factor
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ABSTRACT: Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and melanocytes at resolutions as high as approximately 6 and approximately 20 nm in the directions parallel and perpendicular, respectively, to the direction of ion-beam milling. The 3D images demonstrate the striking spatial relationships between specific organelles such as mitochondria and membranes of the endoplasmic reticulum, and the distribution of unique cellular components such as melanosomes. We also show that 10nm-sized gold particles and quantum dot particles with 7 nm-sized cores can be detected in single cross-sectional images. IA-SEM is thus a useful tool for imaging large mammalian cells in their entirety at resolutions in the nanometer range.
Journal of Structural Biology 01/2009; 166(1):1-7. · 3.41 Impact Factor
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ABSTRACT: The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.
Journal of Biological Chemistry 03/2006; 281(7):4364-70. · 4.77 Impact Factor
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ABSTRACT: Strategies to achieve the highest resolutions in structures of protein complexes determined by cryo-electron microscopy generally involve averaging information from large numbers of individual molecular images. However, significant limitations are posed by heterogeneity in image quality and in protein conformation that are inherent to large data sets of images. Here, we demonstrate that the combination of iterative refinement and stringent molecular sorting is an effective method to obtain substantial improvements in map quality of the 1.8 MDa icosahedral catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus. From a starting set of 42,945 images of the core complex, we show that using only the best 139 particles in the data set produces a map that is superior to those constructed with greater numbers of images, and that the location of many of the alpha-helices in the structure can be unambiguously visualized in a map constructed from as few as 9 particles.
Journal of Structural Biology 09/2004; 147(2):136-45. · 3.41 Impact Factor
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ABSTRACT: Homoepiboxidine (3) and the corresponding N-methyl (4) and N-benzyl (5) derivatives were prepared from a 6beta-carbomethoxynortropane (8). Affinities and functional activities at neuromuscular, central neuronal and ganglionic-type nicotinic receptors were compared to those of epibatidine 1, and epiboxidine 2. Homoepiboxidine had equivalent affinity/activity to epiboxidine at neuromuscular, neuronal alpha4beta2, and most alpha3-containing ganglionic-type nicotinic receptors. The N-substituted derivatives showed reduced affinity/activity at most receptor subtypes. Replacement of the methylisoxazole moiety of 3 and 4 with a methyloxadiazole moiety provided analogues 6 and 7, which had greatly reduced affinity/activity in virtually all assays at nicotinic receptors. Marked analgetic activity in mice occurred at the following ip doses: epibatidine 10 microg/kg; epiboxidine 25 microg/kg; homoepiboxidine 100 microg/kg; N-methylhomoepiboxidine 100 microg/kg; the methyloxadiazole (6) 100 microg/kg. The time course at such ip doses was significantly longer for homoepiboxidine 3 with marked analgesia still manifest at 30 min post-injection. Epiboxidine and the homoepiboxidines were less toxic than epibatidine.
Bioorganic & Medicinal Chemistry 02/2004; 12(1):179-90. · 2.92 Impact Factor
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ABSTRACT: OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 A resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 microm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4A, and electron diffraction patterns show spots to about 3.2 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22(1)2(1) symmetry group. However, the unit cell dimensions of 102.7A x 67.3 A are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3A x 79.0 A. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.
Journal of Structural Biology 01/2004; 144(3):320-6. · 3.41 Impact Factor
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ABSTRACT: We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1). The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.
Journal of Structural Biology 09/2003; 143(2):135-44. · 3.41 Impact Factor
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ABSTRACT: 1. Caffeine at 0.3-10 mM enhanced the binding of [3H]ryanodine to calcium-release channels of rabbit muscle sarcoplasmic reticulum. A variety of other xanthines were as efficacious as caffeine or nearly so, but none appeared markedly more potent. 2. Caffeine at 1 mM markedly inhibited binding of [3H]diazepam to GABAA receptors in rat cerebral cortical membranes. 3. Other xanthines also inhibited binding with certain dimethylpropargylxanthines being nearly fivefold more potent than caffeine. 4. Caffeine at 1 mM stimulated binding of [35S]TBPS to GABAA receptors as did certain other xanthines. 5. The dimethylpropargylxanthines had little effect. 1,3-Dipropyl-8-cyclopentylxanthine at 100 microM had no effect on [3H]diazepam binding, but markedly inhibited [35S]TBPS binding. 6. Structure-activity relationships for xanthines do differ for calcium-release channels and and for different sites on GABAA receptors, but no highly selective lead compounds were identified.
Cellular and Molecular Neurobiology 07/2003; 23(3):331-47. · 1.97 Impact Factor
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ABSTRACT: Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.
The EMBO Journal 12/2002; 21(21):5587-98. · 9.20 Impact Factor
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[show abstract]
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ABSTRACT: Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 Å structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.
The EMBO Journal 10/2002; 21(21):5587-5598. · 9.20 Impact Factor
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ABSTRACT: The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 A resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.
Natural Structural Biology 09/2002; 9(8):597-600.
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ABSTRACT: The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane 'carrier' proteins. OxlT, a representative example of the MFS, is an oxalate-transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 Å resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two-fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.
The EMBO Journal 08/2001; 20(16):4408-4413. · 9.20 Impact Factor
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ABSTRACT: Riboflavin inhibited binding of both agonist and antagonist radioligands to rat brain A(1)-adenosine receptors with K(i) values of approximately 10 µM. In an adenylate cyclase assay with membrane preparations from either rat adipocytes or DDT MF-2 cells, both of which contain A(1)-adenosine receptors, riboflavin inhibited isoproterenol-stimulated cyclase activity with an IC(50) of approximately 20 µM. However, the inhibition of cyclase by riboflavin was not reversed by an A(1)-selective antagonist, nor by pretreatment with pertussis toxin. Thus, neither A(1)-receptors nor G(i)-proteins appear critically involved in the inhibition of cyclase by riboflavin. Riboflavin did block the stimulation by an adenosine analog of [(35)S]GTPγS binding in rat cerebral cortical membranes. However, riboflavin also inhibited the stimulation by fMLP of [(35)S]GTPγS binding in HL-60 cell membranes. Riboflavin inhibited forskolin-stimulated cyclase in membranes from DDT MF-2 cells > rat adipocytes > PC12 cells, hamster CHO M2 cells, and wild-type S49 cells. There was virtually no inhibition of forskolin-stimulated cyclase in membranes of human platelets, rat cerebral cortex, or cyc(-)S49 cells lacking G(s)-proteins. The calcium-stimulated cyclase in rat cerebral cortical membranes was inhibited by riboflavin. A preincubation of membranes with riboflavin markedly enhanced the inhibition for DDT MF-2 and wild-type and cyc(-)S49 membranes. The extent of inhibition in the different cell lines was dependent on the agent used to stimulate cyclase. Riboflavin, like the P-site inhibitor 2´,5´-dideoxyadenosine, was more potent and efficacious when manganese instead of forskolin was used as the stimulant. However, unlike the P-site inhibitor, riboflavin did not markedly inhibit GppNHp- or fluoride-stimulated cyclase. Riboflavin at low micromolar concentrations appears to have three possibly interrelated effects on second messenger systems subserved by G-proteins. These are antagonism at A(1)-adenosine receptors, inhibition of turnover of guanyl nucleotides at G-proteins, and inhibition of adenylate cyclase.
Drug Development Research 10/1997; 42(2):98-108. · 1.19 Impact Factor
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[show abstract]
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ABSTRACT: OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 Å resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 μm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4 Å, and electron diffraction patterns show spots to about 3.2 Å resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22121 symmetry group. However, the unit cell dimensions of 102.7 Å × 67.3 Å are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3 Å × 79.0 Å. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.
Journal of Structural Biology.
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ABSTRACT: The spiropyrrolizidine oximes 236 and 222 and a related spiropyrrolizidine alkaloid, nitropolyzonamine, block nicotinic receptor channels in rat pheochromocytoma PC12 cells and in human medulloblastoma TE671 cells. In PC12 cells with an α3β4(5)-nicotinic receptor, both the spiropyrrolizidine oxime 236 and nitropolyzonamine had ic50 values of about 1.5 μM, while spiropyrrolizidine oxime 222 had an ic50 value of 2.6 μM versus carbamylcholine-elicited sodium-22 influx. In TE671 cells with an α1β1γδ nicotinic receptor, the spiropyrrolizidine oximes 236, 222, and nitropolyzonamine had ic50values of 9.5, 14, and 67 μM, respectively. The inhibitions by the spiropyrrolizidine oxime 236 and nitropolyzonamine appeared to be noncompetitive in nature in both cell lines. In rat cerebral cortical membranes, binding of [3H]nicotine to α4β2 nicotinic receptors was not inhibited significantly by 10 μM concentrations of the spiropyrrolizidine oxime 236, or by nitropolyzonamine, as expected for a noncompetitive blocker. Both compounds at 10 μM had marginal effects on a variety of central receptors, but did inhibit binding of [3H]1,3-di(2-tolyl)guanidine to sigma receptors in mouse brain membranes with ic50values of about 0.5 μM. The spiropyrrolizidine oxime 236 at 10 μM had no effect on batrachotoxin-elicited sodium influx in guinea pig cerebral cortical synaptoneurosomes or on ATP-elicited calcium influx in PC12 cells. Such spiropyrrolizidines represent a new structural class of blockers of nicotinic receptor channels with selectivity for ganglionic-type receptors.
Biochemical Pharmacology.
