Structure and mechanism of the mammalian fructose transporter GLUT5

Nomura, Norimichi; Verdon, Grégory; Kang, Hae Joo; Shimamura, Tatsuro; Sonoda, Yo; Hussien, Saba Abdul; Qureshi, Aziz Abdul; Coincon, M.; Sato, Yuichi; Abe, Hideki; Nakada-Nakura, Yoshiko; Hino, Tsuneo; Arakawa, T.; Kusano-Arai, Osamu; Iwanari, Hiroko; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Kasahara, Michihiro; Iwata, So; Drew, David

doi:10.1038/nature14909

Cited by 217 publications

(402 citation statements)

References 55 publications

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“…For the mammalian glucose transporters, recent progress has been made in solving the crystal structures of several isoforms in both inward and outward conformations (28)(29)(30). Furthermore, our prior work has provided evidence for the binding pocket that mediates transporter inhibition by HIV protease inhibitors (12,20,31).…”

Section: Discussionmentioning

confidence: 99%

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

Kraft

Heitmeier

Putanko

et al. 2016

Antimicrob Agents Chemother

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The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z= factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC 50 ) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening. Malaria is a major threat to the human population in large areas of the world, affecting over 200 million people per year (1, 2). Beyond the effects of this disease on human life, malaria also cripples economic development and burdens the health care systems of countries where malaria is endemic (3). The emergence of parasites with resistance to even the most potent existing antimalarial drugs, such as the artemisinins (4), has made paramount the development of novel drugs that target essential pathways for parasite survival. Blood stage parasites utilize glucose for both biomass production and ATP synthesis (5). The malarial hexose transporter, Plasmodium falciparum hexose transporter (PfHT), first cloned by Woodrow et al. in 1999 (6), mediates parasite transport of glucose, fructose, mannose, and galactose (7). Since PfHT is essential for parasite survival (8), this protein is a highly promising molecular target for antimalarial drug development. This is supported by the ability of compound 3361, a glucose analogue that inhibits PfHT with high selectivity over the human orthologue GLUT1, to inhibit asexual intraerythrocytic growth in culture (9). Compound 3361 is also active against Plasmodium berghei liver and transmission stage parasites in infected mice (10), suggesting that PfHT is a promising target for full life cycle activity. However, while compound 3361 validates efforts to target PfHT, this compound is not itself considered drug-like and is therefore not a valid candidate for lead development (11). We have recently extended these earlier find...

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Section: Discussionmentioning

confidence: 99%

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

Kraft

Heitmeier

Putanko

et al. 2016

Antimicrob Agents Chemother

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“…20 These Glut5 transporters share 88% sequence identity with each other and 13% sequence identity to PCFT, similar to the level of identity of the glycerol-3-phosphate proton symporter previously used for homology modeling of PCFT. 11,21 The resulting models were found to have comparable quality.…”

Section: Homology Modelsmentioning

confidence: 87%

“…20 On the basis of these structures, we built a homology model for PCFT in these 2 conformations. 18 In Figure 7, the 11th transmembrane helix in the inward open conformation, along with the Asn411 residue, are highlighted in blue.…”

Section: Analysis Of a Homology Model Of Pcft In The Inward And Outwamentioning

confidence: 99%

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

et al. 2018

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Key Points• An N411K mutation in the external gate of the proton-coupled folate transporter within the aqueous channel results in impaired function.• The N411K mutation produces a substratespecific defect in transport, resulting in hereditary folate malabsorption.Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates.The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine . lysine . histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated.Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.

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“…In GLUT-like uniporters and, in a more general sense, transporters of the major facilitator superfamily (MFS), DG E may be provided by conserved motif-A-like structural elements stabilizing either the C Out or C In state. 2,4,6,8 However, it should be noted that crystal structures of transporters are determined in the absence of membrane environment but the presence of detergent(s) and crystal packing. Thus, conformations of transporters observed in crystals do not necessarily represent the true resting states of the corresponding proteins in a membrane environment.…”

Section: Thermodynamics Of the Chemical Potential Driven Transportmentioning

confidence: 99%

How does the chemical potential of the substrate drive a uniporter?

Zhang

Han

2016

Protein Science

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Uniporters are a large class of transporters mediating facilitated diffusion of substrates along the direction of the substrate concentration gradient. Recently, structures of several important uniporters have been reported; however, the precise mechanisms of uniporter function remain subject of debate. Here, we present a series of general thermodynamic descriptions of uniporters, aimed at understanding the structure-function relationship of uniporters, and in particular to reconcile biochemical phenomena of uniporters with our previously proposed thermodynamic model of general transporters.Keywords: uniporters; differential binding energy; elastic conformational energy; transition-state energy barrier; rate-limiting step; kinetic asymmetry UniporterThe cellular membrane presents a major energy barrier to hydrophilic small molecules to move across. This barrier is usually so high that translocation of these small molecules across the membrane along their concentration gradients is practically halted, even in the presence of a thermodynamically favorable chemical potential. The function of uniporters is to lower this energy barrier for selected substrates, so that the substrates can be transported along their own concentration gradients, a process called facilitated diffusion. A uniporter is a transporter that does not require energy input other than the substrate chemical potential. Like all transporters, a uniporter differs from a channel in that the former uses the alternating access mechanism 1 while the latter does not. Using this mechanism, a uniporter switches between two major conformations, namely inward-facing (C In ) and outwardfacing (C Out ), allowing its substrate-binding site to be accessible alternatingly to both sides of the membrane. The energy barrier for a hydrophilic molecule to transport passively across the cellular membrane is analogous to the transition-state energy barrier in a chemical reaction. According to the transitionstate theory of enzymology, an enzyme stabilizes the transition state of the reactants, thus effectively lowering the transition-state energy barrier. However, the reaction is driven by the chemical energy Abbreviations: C In , inward-facing conformation; C Out , outwardfacing conformation.

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Structure and mechanism of the mammalian fructose transporter GLUT5

Cited by 217 publications

References 55 publications

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

How does the chemical potential of the substrate drive a uniporter?

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