A three-pocket model for substrate coordination and selectivity by the nucleotide sugar transporters SLC35A1 and SLC35A2.

The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities. Mutations in either induce congenital disorders of glycosylation. Despite the biomedical relevance, mechanisms of substrate specificity are unclear. To address this critical issue, we utilized a structure-guided mutagenesis strategy and assayed a series of SLC35A2 and SLC35A1 mutants using a rescue approach. Our results suggest that three pockets in the central cavity of each transporter provide substrate specificity. The pockets comprise (1) nucleobase (residues E52, K55, and Y214 of SLC35A1; E75, K78, N235, and G239 of SLC35A2); (2) middle (residues Q101, N102, and T260 of SLC35A1; Q125, N126, Q129, Y130, and Q278 of SLC35A2); and (3) sugar (residues K124, T128, S188, and K272 of SLC35A1; K148, T152, S213, and K297 of SLC35A2) pockets. Within these pockets, two components appear to be especially critical for substrate specificity. Y214 (for SLC35A1) and G239 (for SLC35A2) in the nucleobase pocket appear to discriminate cytosine from uracil. Furthermore, Q129 and Q278 of SLC35A2 in the middle pocket appear to interact specifically with the ß-phosphate of UDP while the corresponding A105 and A253 residues in SLC35A1 do not interact with CMP, which lacks a ß-phosphate. Overall, our findings contribute to a molecular understanding of substrate specificity and coordination in SLC35A1 and SLC35A2 and have important implications for the understanding and treatment of diseases associated with mutations or dysregulations of these two transporters.

Orthosteric-allosteric dual inhibitors of PfHT1 as selective antimalarial agents.

Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a "selective starvation" strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure-activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum.

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 and fluorescein-labeled A8-35.

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.

Entropically driven self-assembly of Lysinibacillus sphaericus S-layer proteins analyzed under various environmental conditions.

S-Layer proteins are an example of bionanostructures that can be exploited in nanofabrication. In addition to their ordered structure, the ability to self-assembly is a key feature that makes them a promising technological tool. Here, in vitro self-assembly kinetics of SpbA was investigated, and found that it occurs at a rate that is dependent on temperature, its concentration, and the concentration of calcium ions and sodium chloride. The activation enthalpy (120.81 kJ . mol(-1)) and entropy (129.34 J . mol(-1) . K(-1)) obtained infers that the incorporation of monomers incurs in a net loss of hydrophobic surface. By understanding how the protein monomers drive the self-assembly at different conditions, the rational optimization of this process was feasible.

The prototypical H+/galactose symporter GalP assembles into functional trimers.

Glucose is a primary source of energy for human cells. Glucose transporters form specialized membrane channels for the transport of sugars into and out of cells. Galactose permease (GalP) is the closest bacterial homolog of human facilitated glucose transporters. Here, we report the functional reconstitution and 2D crystallization of GalP. Single particle electron microscopy analysis of purified GalP shows that the protein assembles as an oligomer with three distinct densities. Reconstitution assays yield 2D GalP crystals that exhibit a hexagonal array having p3 symmetry. The projection structure of GalP at 18 A resolution shows that the protein is trimeric. Each monomer in the trimer forms its own channel, but an additional cavity (10 approximately 15 A in diameter) is apparent at the 3-fold axis of the oligomer. We show that the crystalline GalP is able to selectively bind substrate, suggesting that the trimeric form is biologically active.

NMR assignments of the periplasmic loop P2 of the MalF subunit of the maltose ATP binding cassette transporter.

We have assigned the (1)H, (15)N, (13)C backbone resonances of the second periplasmic loop P2 of the MalF subunit of the maltose ATP binding cassette transporter of Escherichia coli/Salmonella which is important for the recognition of the maltose binding protein MalE.

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals.

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.

Bacterial protein patterning by micro-contact printing of PLL-g-PEG.

Biomimetic micro-patterned surfaces of three S-layer (fusion) proteins, wild type (SbpA), enhanced green fluorescence protein (SbpA-EGFP) and streptavidin (SbpA-STV), were built by microcontact printing of poly-L-lysine grafted polyethylene glycol (PLL-g-PEG). The functionality of the adsorbed proteins was studied with atomic force microscopy and fluorescence microscopy. Atomic force microscopy (AFM) measurements showed that wild-type SbpA recrystallized on PLL-g-PEG free areas, while fluorescent properties of SbpA-EGFP and the interaction of SbpA-streptavidin heterotetramers with biotin were not affected due to the adsorption on the micro patterned substrates.

Sugar transport across lactose permease probed by steered molecular dynamics.

Escherichia coli lactose permease (LacY) transports sugar across the inner membrane of the bacterium using the proton motive force to accumulate sugar in the cytosol. We have probed lactose conduction across LacY using steered molecular dynamics, permitting us to follow molecular and energetic details of lactose interaction with the lumen of LacY during its permeation. Lactose induces a widening of the narrowest parts of the channel during permeation, the widening being largest within the periplasmic half-channel. During permeation, the water-filled lumen of LacY only partially hydrates lactose, forcing it to interact with channel lining residues. Lactose forms a multitude of direct sugar-channel hydrogen bonds, predominantly with residues of the flexible N-domain, which is known to contribute a major part of LacY's affinity for lactose. In the periplasmic half-channel lactose predominantly interacts with hydrophobic channel lining residues, whereas in the cytoplasmic half-channel key protein-substrate interactions are mediated by ionic residues. A major energy barrier against transport is found within a tight segment of the periplasmic half-channel where sugar hydration is minimal and protein-sugar interaction maximal. Upon unbinding from the binding pocket, lactose undergoes a rotation to permeate either half-channel with its long axis aligned parallel to the channel axis. The results hint at the possibility of a transport mechanism, in which lactose permeates LacY through a narrow periplasmic half-channel and a wide cytoplasmic half-channel, the opening of which is controlled by changes in protonation states of key protein side groups.

Lactose permease H+-lactose symporter: mechanical switch or Brownian ratchet?

Lactose permease structure is deemed consistent with a mechanical switch device for H(+)-coupled symport. Because the crystallography-assigned docking position of thiodigalactoside (TDG) does not make close contact with several amino acids essential for symport; the switch model requires allosteric interactions between the proton and sugar binding sites. The docking program, Autodock 3 reveals other lactose-docking sites. An alternative cotransport mechanism is proposed where His-322 imidazolium, positioned in the central pore equidistant (5-7 A) between six charged amino acids, Arg-302 and Lys-319 opposing Glu-269, Glu-325, Asp-237, and Asp-240, transfers a proton transiently to an H-bonded lactose hydroxyl group. Protonated lactose and its dissociation product H(3)O+ are repelled by reprotonated His-322 and drift in the electrostatic field toward the cytosol. This Brownian ratchet model, unlike the conventional carrier model, accounts for diminished symport by H322N mutant; how H322 mutants become uniporters; why exchanging Lys-319 with Asp-240 paradoxically inactivates symport; how some multiple mutants become revertant transporters; the raised export rate and affinity toward lactose of uncoupled mutants; the altered specificity toward lactose, melibiose, and galactose of some mutants, and the proton dissociation rate of H322 being 100-fold faster than the symport turnover rate.

Degradation of the hexose transporter Hxt5p in Saccharomyces cerevisiae.

BACKGROUND INFORMATION: Hxt5p is a member of a multigene family of hexose transporter proteins which translocate glucose across the plasma membrane of the yeast Saccharomyces cerevisiae. In contrast with other major hexose transporters of this family, Hxt5p expression is regulated by the growth rate of the cells and not by the external glucose concentration. Furthermore, Hxt5p is the only glucose transporter expressed during stationary phase. These observations suggest a different role for Hxt5p in S. cerevisiae. Therefore we studied the metabolism and localization of Hxt5p in more detail. RESULTS AND CONCLUSIONS: Inhibition of HXT5 expression in stationary-phase cells by the addition of glucose, which increases the growth rate, led to a decrease in the amount of Hxt5 protein within a few hours. Addition of glucose to stationary-phase cells resulted in a transient phosphorylation of Hxt5p on serine residues, but no ubiquitination was detected. The decrease in Hxt5p levels is caused by internalization of the protein, as observed by immunofluorescence microscopy. In stationary-phase cells, Hxt5p was localized predominantly at the cell periphery and upon addition of glucose to the cells the protein translocated to the cell interior. Electron microscopy demonstrated that the internalized Hxt5p-HA (haemagglutinin) protein was localized to small vesicles, multivesicular bodies and the vacuole. These results suggest that internalization and degradation of Hxt5p in the vacuole occur in an ubiquitination-independent manner via the endocytic pathway.

Structure, surface interactions, and compressibility of bacterial S-layers through scanning force microscopy and the surface force apparatus.

Two-dimensional crystalline bacterial surface layers (S-layers) are found in a broad range of bacteria and archaea as the outermost cell envelope component. The self-assembling properties of the S-layers permit them to recrystallize on solid substrates. Beyond their biological interest as S-layers, they are currently used in nanotechnology to build supramolecular structures. Here, the structure of S-layers and the interactions between them are studied through surface force techniques. Scanning force microscopy has been used to study the structure of recrystallized S-layers from Bacillus sphaericus on mica at different 1:1 electrolyte concentrations. They give evidence of the two-dimensional organization of the proteins and reveal small corrugations of the S-layers formed on mica. The lattice parameters of the S-layers were a=b=14 nm, gamma=90 degrees and did not depend on the electrolyte concentration. The interaction forces between recrystallized S-layers on mica were studied with the surface force apparatus as a function of electrolyte concentration. Force measurements show that electrostatic and steric interactions are dominant at long distances. When the S-layers are compressed they exhibit elastic behavior. No adhesion between recrystallized layers takes place. We report for the first time, to our knowledge, the value of the compressibility modulus of the S-layer (0.6 MPa). The compressibility modulus is independent on the electrolyte concentration, although loads of 20 mN m-1 damage the layer locally. Control experiments with denatured S-proteins show similar elastic properties under compression but they exhibit adhesion forces between proteins, which were not observed in recrystallized S-layers.

Atomic force microscopy study of Escherichia coli lactose permease proteolipid sheets.

Proteolipid sheets (PLSs) obtained using the vesicle fusion technique on a convenient surface are the base to obtain transmembrane protein biosensors. In this preliminary work, we have screened several physicochemical conditions to optimize the visualization of proteolipid sheets formed between different phospholipid matrices and the membrane protein lactose permease (LacP) by atomic force microscopy (AFM). When LacP was reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, the proteolipid sheets were densely packed with an upper layer that protruded from a background layer. Several lipid protein molar ratios (LPR) were screened. High resolution analysis of the upper layer revealed a quasi-crystalline arrangement formed by small entities that could be attributed to the protein. The approach described here may be suitable for the rational design of biosensors based in other transmembrane proteins.

Coupled sodium/glucose cotransport by SGLT1 requires a negative charge at position 454.

Na(+)/glucose cotransport by SGLT1 is a tightly coupled process that is driven by the Na(+) electrochemical gradient across the plasma membrane. We have previously proposed that SGLT1 contains separate Na(+)- and glucose-binding domains, that A166 (in the Na(+) domain) is close to D454 (in the sugar domain), and that interactions between these residues influence sugar specificity and transport. We have now expressed the mutant D454C in Xenopus laevis oocytes and examined the role of charge on residue 454 by replacing the Asp with Cys or His, and by chemical mutation of D454C with alkylating reagents of different charge (MTSES(-), MTSET(+), MMTS(0), MTSHE(0), and iodoacetate(-)). Functional properties were examined by measuring sugar transport and cotransporter currents. In addition, D454C was labeled with fluorescent dyes and the fluorescence of the labeled transporter was recorded as a function of voltage and ligand concentration. The data shows that (1) aspartate 454 is critically important for the normal trafficking of the protein to the plasma membrane; (2) there were marked changes in the functional properties of D454C, i.e., a reduction in turnover number and a loss of voltage sensitivity, although there were no alterations in sugar selectivity or sugar and Na(+) affinity; (3) a negative charge on residue 454 increased Na(+) and sugar transport with a normal stoichiometry of 2 Na(+):1 sugar. A positive charge on residue 454, in contrast, uncoupled Na(+) and sugar transport, indicating the importance of the negative charge in the coordination of the cotransport mechanism.

Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain--an immunohistochemical study.

The aim of this work was to study the distribution and cellular localization of GLUT2 in the rat brain by light and electron microscopic immunohistochemistry, whereas our ultrastructural observations will be reported in a second paper. Confirming previous results, we show that GLUT2-immunoreactive profiles are present throughout the brain, especially in the limbic areas and related nuclei, whereas they appear most concentrated in the ventral and medial regions close to the midline. Using cresyl violet counterstaining and double immunohistochemical staining for glial or neuronal markers (GFAp, CAII and NeuN), we show that two limited populations of oligodendrocytes and astrocytes cell bodies and processes are immunoreactive for GLUT2, whereas a cross-reaction with GLUT1 cannot be ruled out. In addition, we report that the nerve cell bodies clearly immunostained for GLUT2 were scarce (although numerous in the dentate gyrus granular layer in particular), whereas the periphery of numerous nerve cells appeared labeled for this transporter. The latter were clustered in the dorsal endopiriform nucleus and neighboring temporal and perirhinal cortex, in the dorsal amygdaloid region, and in the paraventricular and reuniens thalamic nuclei, whereas they were only a few in the hypothalamus. Moreover, a group of GLUT2-immunoreactive nerve cell bodies was localized in the dorsal medulla oblongata while some large multipolar nerve cell bodies peripherally labeled for GLUT2 were scattered in the caudal ventral reticular formation. This anatomical localization of GLUT2 appears characteristic and different from that reported for the neuronal transporter GLUT3 and GLUT4. Indeed, the possibility that GLUT2 may be localized in the sub-plasmalemnal region of neurones and/or in afferent nerve fibres remains to be confirmed by ultrastructural observations. Because of the neuronal localization of GLUT2, and of its distribution relatively similar to glucokinase, it may be hypothesized that this transporter is, at least partially, involved in cerebral glucose sensing.

Immunocytochemical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. II. Electron microscopic study.

Following a former immunohistochemical study in the rat brain [Arluison, M., Quignon, M., Nguyen, P., Thorens, B., Leloup, C., Penicaud, L. Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. I. Immunohistochemical study. J. Chem. Neuroanat., in press], we have analyzed the ultrastructural localization of GLUT2 in representative and/or critical areas of the forebrain and hindbrain. In agreement with previous results, we observe few oligodendrocyte and astrocyte cell bodies discretely labeled for GLUT2 in large myelinated fibre bundles and most brain areas examined, whereas the reactive glial processes are more numerous and often localized in the vicinity of nerve terminals and/or dendrites or dendritic spines forming synaptic contacts. Only some of them appear closely bound to unlabeled nerve cell bodies and dendrites. Furthermore, the nerve cell bodies prominently immunostained for GLUT2 are scarce in the brain nuclei examined, whereas the labeled dendrites and dendritic spines are relatively numerous and frequently engaged in synaptic junctions. In conformity with the observation of GLUT2-immunoreactive rings at the periphery of numerous nerve cell bodies in various brain areas (see previous paper), we report here that some neuronal perikarya of the dorsal endopiriform nucleus/perirhinal cortex exhibit some patches of immunostaining just below the plasma membrane. However, the presence of many GLUT2-immunoreactive nerve terminals and/or astrocyte processes, some of them being occasionally attached to nerve cell bodies and dendrites, could also explain the pericellular labeling observed. The results here reported support the idea that GLUT2 may be expressed by some cerebral neurones possibly involved in glucose sensing, as previously discussed. However, it is also possible that this transporter participate in the regulation of neurotransmitter release and, perhaps, in the release of glucose by glial cells.

Direct imaging of lateral movements of AMPA receptors inside synapses.

Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses.

Peripheral glucose administration stimulates the translocation of GLUT8 glucose transporter to the endoplasmic reticulum in the rat hippocampus.

The expression and localization of glucose transporter isoforms play essential roles in the glucoregulatory activities of the hippocampus and ultimately contribute to cognitive status in physiological and pathophysiological settings. The recently identified glucose transporter GLUT8 is uniquely expressed in neuronal cell bodies in the rat hippocampus and therefore may contribute to hippocampal glucoregulatory activities. We show here that GLUT8 has a novel intracellular distribution in hippocampal neurons and is translocated to intracellular membranes following glucose challenge. Immunoblot analysis revealed that GLUT8 is expressed in high-density microsomes (HDM), suggesting that GLUT8 is associated with intracellular organelles under basal conditions. Immunogold electron microscopic analysis confirmed this observation, in that GLUT8 immunogold particles were associated with the rough endoplasmic reticulum (ER) and cytoplasm. Peripheral glucose administration produced a rapid twofold increase in GLUT8 levels in the HDM fraction while decreasing GLUT8 levels in low-density microsomes. Similarly, peripheral glucose administration significantly increased GLUT8 association with the rough ER in the hippocampus. Conversely, under hyperglycemic/insulinopenic conditions, namely, in streptozotocin (STZ) diabetes, hippocampal GLUT8 protein levels were decreased in the HDM fraction. These results demonstrate that GLUT8 undergoes rapid translocation to the rough ER in the rat hippocampus following peripheral glucose administration, trafficking that is impaired in STZ diabetes, suggesting that insulin serves as a stimulus for GLUT8 translocation in hippocampal neurons. Because glucose is liberated from oligosaccharides during N-linked glycosylation events in the rough ER, we propose that GLUT8 may serve to transport glucose out of the rough ER into the cytosol and in this manner contribute to glucose homeostasis in hippocampal neurons.