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Christine Ziegler - One of the best experts on this subject based on the ideXlab platform.
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Carr–Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses
2015Co-Authors: Philipp E. Spindler, Christine Ziegler, Izabela Waclawska, Burkhard Endeward, Jörn Plackmeyer, Thomas F. PrisnerAbstract:Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5–8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr–Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric Betaine Transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods
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Substrate-bound outward-open state of the Betaine Transporter BetP provides insights into Na^+ coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Lucy R Forrest, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. Francesconi, Christine ZieglerAbstract:The Na^+-coupled Betaine Transporter BetP is representative of a structural superfamily of symporters, for which different conformational states of the transport cycle are described. Perez et al. provide a structure for the elusive substrate-bound outward-open state, and propose a mechanism for sodium-coupled transport. The Na^+-coupled Betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary Transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary Transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of Betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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substrate bound outward open state of the Betaine Transporter betp provides insights into na coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Lucy R Forrest, Christine Ziegler, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. FrancesconiAbstract:The Na(+)-coupled Betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary Transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary Transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of Betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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lock and load key role of the unique closed state in transport regulation of the sodium coupled Betaine Transporter betp
Biophysical Journal, 2014Co-Authors: Caroline Koshy, Izabela Waclawska, Christine ZieglerAbstract:The Na+-coupled Betaine symporter BetP is regulated by osmotic stress sensing the increasing internal K+ concentration as a direct consequence of hypertonicity by its positively charged C-terminal domain. Recently, we demonstrated that BetP binds K+ cooperatively to its cytoplasmic side between different C-terminal domains within the BetP trimer, while the monomer is not regulated. K+ binding strengthens the helical fold of the C-terminal domain, and together with negatively charged lipids at the trimer center orients it towards the counterclockwise adjacent protomer. The extended C-terminal conformation is assisted by an additional interaction with the N-terminal domain and most crucially with lipids. A recent crystal structure show BetP in a closed state with the C-terminal domain bending back towards its own protomer to interact mainly with lipid head groups at the membrane surface. Thereby, an intratrimeric interaction is inhibited, which allows us to assign the bend-back conformation of the C-terminal domain as the inactive one and the extended conformation as active. A surprising consequence of the inactive C-terminal conformation is the uncoupling of the two sodium sites. Although having all substrates bound BetP cannot any longer isomerize to the inward-facing state because the C-terminal domain together with lipids lock the intracellular gate preventing the opening of the S1 site. Subsequently, all BetP protomers within a trimer remain fully substrate loaded in a closed state with additional Betaine molecules sequestered and stored in the second periplasmic (S2) binding site. We propose a trimeric switch model, in which osmotic stress induces a stepwise activation of one protomer after the other by consecutive K+ binding and straightening of the C-terminal helix.
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structural evidence for functional lipid interactions in the Betaine Transporter betp
The EMBO Journal, 2013Co-Authors: Caroline Koshy, Camilo Perez, Christine Ziegler, Eva S Schweikhard, Rebecca M Gartner, Ozkan YildizAbstract:Bilayer lipids contribute to the stability of membrane Transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or Transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated Betaine Transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid–protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport.
Camilo Perez - One of the best experts on this subject based on the ideXlab platform.
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substrate bound outward open state of the Betaine Transporter betp provides insights into na coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Lucy R Forrest, Christine Ziegler, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. FrancesconiAbstract:The Na(+)-coupled Betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary Transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary Transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of Betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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Substrate-bound outward-open state of the Betaine Transporter BetP provides insights into Na^+ coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Lucy R Forrest, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. Francesconi, Christine ZieglerAbstract:The Na^+-coupled Betaine Transporter BetP is representative of a structural superfamily of symporters, for which different conformational states of the transport cycle are described. Perez et al. provide a structure for the elusive substrate-bound outward-open state, and propose a mechanism for sodium-coupled transport. The Na^+-coupled Betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary Transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary Transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of Betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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the Betaine Transporter betp analysis of osmotic stimuli responsible for activation
Biophysical Journal, 2014Co-Authors: Stanislaw Maximov, Camilo Perez, Becker Markus, Ziegler Christine, Reinhard KraemerAbstract:The Na+ coupled Betaine uptake system BetP of Corynebacterium glutamicum is an established model system for osmoregulated membrane Transporters in bacteria. It belongs to the BCCT family of Transporters and comprises both a catalytic function (Betaine/Na+ cotransport) and a sensory/regulatory function (responding to osmotic stress).Its 2D (electron crystallography) and 3D structure (X-ray crystallography) has been solved. Within a homooligomeric trimer, each BetP protomer harbours both an N- and a C-terminal domain involved in stimulus sensing and intramolecular signal transduction. Factors known so far contributing to the sensory and regulatory function of BetP are (i) the two terminal domains, (ii) K+ ions as an osmostress related stimulus, and (iii) interaction with the surrounding membrane. The analysis of relevant stimuli related to osmotic activation was previously done mainly in proteoliposomes (reconstituted system).We have now performed a stimulus analysis of BetP in intact cells under different levels of internal K+ and found that, different from the previous conception, the second stimulus is more relevant for BetP activation in response to hyperosmotic stress under physiological conditions. This second stimulus was shown to act on BetP directly via the membrane surrounding. In addition, these results shed new light on differences between an in vivo (intact cells) and in vitro analysis (proteoliposomes) of a well-studied membrane Transporter.
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structural evidence for functional lipid interactions in the Betaine Transporter betp
The EMBO Journal, 2013Co-Authors: Caroline Koshy, Camilo Perez, Christine Ziegler, Eva S Schweikhard, Rebecca M Gartner, Ozkan YildizAbstract:Bilayer lipids contribute to the stability of membrane Transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or Transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated Betaine Transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid–protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport.
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structural evidence for functional lipid interactions in the Betaine Transporter betp
Biophysical Journal, 2013Co-Authors: Caroline Koshy, Camilo Perez, Christine Ziegler, Eva S Schweikhard, Rebecca M Gartner, Ozkan YildizAbstract:Bilayer lipids contribute greatly to the stability of membrane Transporters and are crucially involved in their proper functioning. However, the molecular details of how these lipids affect membrane transport is limited to the few atomic resolution structures of channels and Transporters revealing functional lipid interactions. As a consequence, despite their biological importance, bilayer lipid-protein interactions are rarely considered in the molecular description of a transport mechanism, which are difficult to detect in X-ray structures per se. Lipids are often depleted from the detergent-protein complex during isolation or appear too unordered to identify in the crystal. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated Betaine Transporter BetP in complex with anionic lipids. Activity regulation in BetP depends strongly on the presence of negatively charged lipids. We observe eight fully resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids bound to one BetP trimer, mimicking parts of the membrane leaflets. Our data reveal that the PG lipids interact with key residues in transport and regulation. Lipids, while likely being involved in the correct assembly of the BetP trimer also offer important communication sites between the protomers that might be required during stress sensing and transport regulation. The lipid-protein interactions observed in BetP reiterate the influence that the surrounding membrane can have on membrane protein function and urge a more holistic approach towards understanding membrane transport, especially for LeuT-like fold Transporters.
Reinhard Kramer - One of the best experts on this subject based on the ideXlab platform.
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The SLC6 Transporters: perspectives on structure, functions, regulation, and models for Transporter dysfunction
Pflügers Archiv - European Journal of Physiology, 2014Co-Authors: Gary Rudnick, Reinhard Kramer, Randy D. Blakely, Dennis L. Murphy, Francois VerreyAbstract:The human SLC6 family is composed of approximately 20 structurally related symporters (co-Transporters) that use the transmembrane electrochemical gradient to actively import their substrates into cells. Approximately half of the substrates of these Transporters are amino acids, with others transporting biogenic amines and/or closely related compounds, such as nutrients and compatible osmolytes. In this short review, five leaders in the field discuss a number of currently important research themes that involve SLC6 Transporters, highlighting the integrative role they play across a wide spectrum of different functions. The first essay, by Gary Rudnick, describes the molecular mechanism of their coupled transport which is being progressively better understood based on new crystal structures, functional studies, and modeling. Next, the question of multiple levels of Transporter regulation is discussed by Reinhard Krämer, in the context of osmoregulation and stress response by the related bacterial Betaine Transporter BetP. The role of selected members of the human SLC6 family that function as nutrient amino acid Transporters is then reviewed by François Verrey. He discusses how some of these Transporters mediate the active uptake of (essential) amino acids into epithelial cells of the gut and the kidney tubule to support systemic amino acid requirements, whereas others are expressed in specific cells to support their specialized metabolism and/or growth. The most extensively studied members of the human SLC6 family are neurotransmitter reuptake Transporters, many of which are important drug targets for the treatment of neuropsychiatric disorders. Randy Blakely discusses the role of posttranscriptional modifications of these proteins in regulating Transporter subcellular localization and activity state. Finally, Dennis Murphy reviews how natural gene variants and mouse genetic models display consistent behavioral alterations that relate to altered extracellular neurotransmitter levels.
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conformational changes of the Betaine Transporter betp from corynebacterium glutamicum studied by pulse epr spectroscopy
Biochimica et Biophysica Acta, 2012Co-Authors: Sascha C T Nicklisch, Dorith Wunnicke, I V Borovykh, Susanne Morbach, Johann P Klare, Heinzjurgen Steinhoff, Reinhard KramerAbstract:The Betaine Transporter BetP from Corynebacterium glutamicum is activated by hyperosmotic stress critically depending on the presence and integrity of its sensory C-terminal domain. The conformational properties of the trimeric BetP reconstituted in liposomes in the inactive state and during osmotic activation were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Comparison of intra- and intermolecular inter spin distance distributions obtained by double electron-electron resonance (DEER) EPR with the crystal structure of BetP by means of a rotamer library analysis suggest a rotation of BetP protomers within the trimer by about 15° as compared to the X-ray structure. Furthermore, we observed conformational changes upon activation of BetP, which are reflected in changes of the distances between positions 545 and 589 of different protomers in the trimer. Introduction of proline at positions 550 and 572, both leading to BetP variants with a permanent (low level) transport activity, caused changes of the DEER data similar to those observed for the activated and inactivated state, respectively. This indicates that not only displacements of the C-terminal domain in general but also concomitant interactions of its primary structure with surrounding protein domains and/or lipids are crucial for the activity regulation of BetP.
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the role of trimerization in the osmoregulated Betaine Transporter betp
EMBO Reports, 2011Co-Authors: Camilo Perez, Kamil Khafizov, Lucy R Forrest, Reinhard Kramer, Christine ZieglerAbstract:The osmoregulated Betaine Transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate Betaine, but is no longer regulated by hyperosmotic shock.
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The BCCT-family of carriers: from physiology to crystal structure
Molecular Microbiology, 2010Co-Authors: Christine Maria Ziegler, Erhard Bremer, Reinhard KramerAbstract:To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes. Osmotically controlled uptake systems allow the scavenging of these compounds from environmental sources as osmoprotectants. Some of these Transporters belong to the BCCT family (Betaine-choline-carnitine-Transporter), sodium- or proton-coupled carriers (e.g. BetP and BetT, respectively) that are ubiquitous in microorganisms. The BCCT family also contains CaiT, a L-carnitine/γ-butyroBetaine antiporter that is not involved in osmotic stress responses. The glycine Betaine Transporter BetP from Corynebacterium glutamicum is a representative for osmoregulated symporters of the BCCT-family and functions both as an osmosensor and osmoregulator. The crystal structure of BetP in complex with its substrate glycine Betaine and two crystal structures of CaiT in complex with L-carnitine and γ-butyroBetaine were reported recently. These structures, and biochemical data on the activity control of BetP in response to osmotic stress enables a correlation between the sensing of osmotic stress by a Transporter protein with the ensuing regulation of transport activity. Molecular determinants governing the high-affinity binding of the compatible solutes by BetP and CaiT, the coupling in symporters and antiporters, and the osmoregulatory properties are discussed in detail for BetP and various BCCT carriers.
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regulative interactions of the osmosensing c terminal domain in the trimeric glycine Betaine Transporter betp from corynebacterium glutamicum
Biological Chemistry, 2009Co-Authors: Reinhard Kramer, Christine ZieglerAbstract:Activation of the osmoregulated trimeric Betaine Transporter BetP from Corynebacterium glutamicum was shown to depend mainly on the correct folding and integrity of its 55 amino acid long, partly alpha-helical C-terminal domain. Reorientation of the three C-terminal domains in the BetP trimer indicates different lipid-protein and protein-protein interactions of the C-terminal domain during osmoregulation. A regulation mechanism is suggested where this domain switches the Transporter from the inactive to the active state. Interpretation of recently obtained electron and X-ray crystallography data of BetP led to a structure-function based model of C-terminal molecular switching involved in osmoregulation.
J S Handler - One of the best experts on this subject based on the ideXlab platform.
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The canine sodium/myo-inositol coTransporter gene: Structural organization and characterization of the promoter
'Elsevier BV', 2015Co-Authors: Js Rim, Takenaka M, J S Handler, Tanawattanacharoen S, Kwon H. MooAbstract:The sodium/myo-inositol coTransporter (SMIT) is a plasma membrane protein catalyzing transfer of myoinositol into cells against a considerable concentration gradient using the electrochemical potential of sodium across the cell membrane, Transcription of the SMIT gene is markedly stimulated when cells are exposed to a hypertonic environment resulting in increased abundance of SMIT mRNA and increased SMIT activity, The increased accumulation of myo-inositol protects cells from the deleterious effects of hypertonicity, In an effort toward understanding transcriptional regulation, we cloned canine genomic DNA fragments containing the SMIT gene. The gene is 37 kb in size consisting of 2 exons and a large intron of 25 kb, The entire open reading frame is in the second exon, The promoter of the gene is highly active due to a GC-rich sequence. Ribonuclease protection assay using a riboprobe complementary to the 5' end of the gene confirmed that the promoter of the gene is stimulated by hypertonicity, The promoters and regulatory sequences of the SMIT gene and the Betaine Transporter gene, another gene regulated by hypertonicity, appear to be different.close1
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THE CANINE Betaine GAMMA-AMINO-N-BUTYRIC ACID Transporter GENE - DIVERSE MESSENGER-RNA ISOFORMS ARE REGULATED BY HYPERTONICITY AND ARE EXPRESSED IN A TISSUE-SPECIFIC MANNER
'Proceedings of the National Academy of Sciences', 2015Co-Authors: Takenaka M, Yamauchi A, A S Preston, Kwon H. Moo, Sm Bagnasco, Uchida S, J S HandlerAbstract:The Na+- and Cl--coupled Betaine Transporter, designated BGT1, a member of the neurotransmitter Transporter gene family, is responsible for accumulation of Betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla, The canine gene for the Betaine gamma-amino-n-butyric acid Transporter has been cloned and analyzed, The gene extends over 28 kb and consists of 18 exons, The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D), Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing, Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence, Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells, Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters, Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.open5
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Osmolarity in renal medulla of transgenic mice regulates transcription via 5'-flanking region of canine BGT1 gene.
American Journal of Physiology-Renal Physiology, 1997Co-Authors: Kaneko Tetsuya, H M Kwon, J S Handler, Masaru Takenaka, Masaru Okabe, Y. Yoshimura, Atsushi Yamauchi, Masaru Horio, Enyu ImaiAbstract:Betaine is a major compatible osmolyte accumulated in the mammalian kidney medulla and in Madin-Darby canine kidney cells in response to hypertonicity. The accumulation is the result of an increase in maximal velocity of the Na(+)- and Cl-coupled Betaine Transporter designated BGT1. We have previously cloned the canine BGT1 gene and identified a tonicity-responsive enhancer element (TonE) in its 5'-flanking region. Here we report studies of transgenic mice that have in their genome 2.4 kb of the 5'-flanking region of the canine BGT1 gene in front of a chloramphenicol acetyl-transferase (CAT) reporter. Expression of CAT mRNA was detected only in the renal medulla and was increased by experimental manipulations that increase the tonicity of the renal medulla and decreased by manipulations that decrease medullary tonicity. We conclude that the 2.4-kb 5'-flanking region of the BGT1 gene mediates an increase in transcription in response to hyperosmolarity in the renal medulla.
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the canine Betaine gamma amino n butyric acid Transporter gene diverse mrna isoforms are regulated by hypertonicity and are expressed in a tissue specific manner
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: M Takenaka, H M Kwon, Shinichi Uchida, A Yamauchi, A S Preston, Sm Bagnasco, J S HandlerAbstract:Abstract The Na(+)- and Cl(-)-coupled Betaine Transporter, designated BGT1, a member of the neurotransmitter Transporter gene family, is responsible for accumulation of Betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. The canine gene for the Betaine gamma-amino-n-butyric acid Transporter has been cloned and analyzed. The gene extends over 28 kb and consists of 18 exons. The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D). Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing. Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence. Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells. Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.
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the tonicity sensitive element that mediates increased transcription of the Betaine Transporter gene in response to hypertonic stress
Journal of Biological Chemistry, 1994Co-Authors: M Takenaka, H M Kwon, A S Preston, J S HandlerAbstract:Abstract BGT1, the Na(+)-and Cl- coupled Betaine Transporter, is responsible for the accumulation of high concentrations of the non-perturbing osmolyte Betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. In MDCK cells, the increase in activity of the Betaine Transporter is preceded by an increase in transcription of BGT1 and in the abundance of BGT1 mRNA. To investigate the molecular mechanism of transcriptional regulation by tonicity, we have characterized the 5'-flanking region of the gene. Transient transfection assays in MDCK cells cultured in isotonic or hypertonic medium using luciferase reporter constructs containing various fragments of the 5'-flanking region revealed that the region spanning base pairs -69 to -50 5' to the transcription initiation site (-69/-50) has hypertonicity-responsive enhancer activity. A double-stranded -69/-50 concatemer cloned 5' to an SV40 basal promoter and luciferase reporter gene in hypertonic cells exhibited more than 11-fold the activity in isotonic cells. Expression assays and electrophoretic mobility shift assays of mutants of -69/-50 identified a smaller region that is required for hypertonicity to induce increased expression and a slowly migrating band on mobility shift assays.
Lucy R Forrest - One of the best experts on this subject based on the ideXlab platform.
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Substrate-bound outward-open state of the Betaine Transporter BetP provides insights into Na^+ coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Lucy R Forrest, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. Francesconi, Christine ZieglerAbstract:The Na^+-coupled Betaine Transporter BetP is representative of a structural superfamily of symporters, for which different conformational states of the transport cycle are described. Perez et al. provide a structure for the elusive substrate-bound outward-open state, and propose a mechanism for sodium-coupled transport. The Na^+-coupled Betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary Transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary Transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of Betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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substrate bound outward open state of the Betaine Transporter betp provides insights into na coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Lucy R Forrest, Christine Ziegler, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. FrancesconiAbstract:The Na(+)-coupled Betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary Transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary Transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of Betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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investigation of the sodium binding sites in the sodium coupled Betaine Transporter betp
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Kamil Khafizov, Camilo Perez, Christine Ziegler, Caroline Koshy, Matthias Quick, Klaus Fendler, Lucy R ForrestAbstract:Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled Transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled Betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related Transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent Km and Kd for sodium, as measured by Betaine uptake, tryptophan fluorescence, and 22Na+ binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.
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the role of trimerization in the osmoregulated Betaine Transporter betp
EMBO Reports, 2011Co-Authors: Camilo Perez, Kamil Khafizov, Lucy R Forrest, Reinhard Kramer, Christine ZieglerAbstract:The osmoregulated Betaine Transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate Betaine, but is no longer regulated by hyperosmotic shock.
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computational and experimental studies of substrate binding conformational change and importance of the trimeric state in the glycine Betaine Transporter betp
Biophysical Journal, 2011Co-Authors: Kamil Khafizov, Camilo Perez, Christine Ziegler, Chingju Tsai, Lucy R ForrestAbstract:The glycine Betaine/sodium symporter BetP responds to changes in external osmolality by regulation of its transport activity. A recent X-ray structure of BetP confirms that it is a homotrimer and in this structure each protomer adopts an identical conformation, in which the pathway is occluded from both sides. Despite the availability of a wealth of experimental data for BetP, the structures of the alternate states (e.g., open to the outside of the cell), molecular mechanisms of substrate and Na+ binding and transport, as well as the functional implications of the trimeric state remain poorly understood. To address these questions, we carried out computational studies using a range of techniques to derive hypotheses that were then tested experimentally. First, to identify structural features of the alternate states, we developed a procedure for flexible fitting of the X-ray structure of BetP into a lower-resolution cryo-EM map of BetP in a more native lipid environment, in which the three protomers have different conformations. These results suggest that: (i) the protomers adopt distinct conformational states relevant to the transport cycle; and (ii) there is conformational coupling between the protomers. Second, we performed all-atom molecular dynamics simulations and in silico alanine scanning of BetP trimers in order to identify interface residues crucial for maintaining the trimeric state. Mutations of these residues to alanine were introduced experimentally revealing that the isolated monomers are functional, and that the trimeric state is important for the regulation and higher activity of the protein. Finally, using molecular modeling and biochemical experiments we identified two Na+ binding sites in BetP that could not be resolved in the 3.35 A resolution X-ray structure.