Publications by authors named "Katharina Dürr"

21 Publications

  • Page 1 of 1

Expression Screening of Human Integral Membrane Proteins Using BacMam.

Methods Mol Biol 2021 ;2199:95-115

Structural Genomics Consortium, University of Oxford, Oxford, UK.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.
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http://dx.doi.org/10.1007/978-1-0716-0892-0_6DOI Listing
March 2021

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

Lactococci of Local Origin as Potential Starter Cultures
for Traditional Montenegrin Cheese Production.

Food Technol Biotechnol 2017 Mar;55(1):55-66

BOKU - University of Natural Resources and Life Sciences, Department of Food Science and Technology, Muthgasse 18, AT-1190 Vienna, Austria.

The aim of this study is to characterise and examine the biochemical properties of 40 strains isolated from indigenous Montenegrin dairy products in order to explore their potential to be used as starter cultures for producing typical Montenegrin cheese, such as 'bijeli sir', 'masni sir' and 'njeguški sir'. Their safety regarding the production of biogenic amines, the presence of antimicrobial resistance and the antibacterial activity against relevant pathogens and spoilage microorganisms has also been tested. Based on the characterisation, all strains belong to ssp. . Out of these 40 strains, 23 displayed rapid acidification ability and proteolysis. However, none of the strains exhibited the ability of lipid degradation. Most of the strains were not associated with any health risk investigated. Summing up, a large percentage (27.5%) of the tested strains showed good properties. These strains should be further examined for their possible application as specific starter cultures in the production of indigenous cheese in Montenegro.
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http://dx.doi.org/10.17113/ftb.55.01.17.4854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434366PMC
March 2017

Reactions of Superoxide with Iron Porphyrins in the Bulk and the Near-Surface Region of Ionic Liquids.

Inorg Chem 2015 Jul 9;54(14):6862-72. Epub 2015 Jul 9.

†Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

The redox reaction of superoxide (KO2) with highly charged iron porphyrins (Fe(P4+), Fe(P8+), and Fe(P8-)) has been investigated in the ionic liquids (IL) [EMIM][Tf2N] (1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide) and [EMIM][B(CN)4] (1-ethyl-3-methylimidazolium tetracyanoborate) by using time-resolved UV/vis stopped-flow, electrochemistry, cryospray mass spectrometry, EPR, and XPS measurements. Stable KO2 solutions in [EMIM][Tf2N] can be prepared up to a 15 mM concentration and are characterized by a signal in EPR spectrum at g = 2.0039 and by the 1215 cm(-1) stretching vibration in the resonance Raman spectrum. While the negatively charged iron porphyrin Fe(P8-) does not react with superoxide in IL, Fe(P4+) and Fe(P8+) do react in a two-step process (first a reduction of the Fe(III) to the Fe(II) form, followed by the binding of superoxide to Fe(II)). In the reaction with KO2, Fe(P4+) and Fe(P8+) show similar rate constants (e.g., in the case of Fe(P4+): k1 = 18.6 ± 0.5 M(-1) s(-1) for the first reaction step, and k2 = 2.8 ± 0.1 M(-1) s(-1) for the second reaction step). Notably, these rate constants are four to five orders of magnitude lower in [EMIM][Tf2N] than in conventional solvents such as DMSO. The influence of the ionic liquid is also apparent during electrochemical experiments, where the redox potentials for the corresponding Fe(III)/Fe(II) couples are much more negative in [EMIM][Tf2N] than in DMSO. This modified redox and kinetic behavior of the positively charged iron porphyrins results from their interactions with the anions of the ionic liquid, while the nucleophilicity of the superoxide is reduced by its interactions with the cations of the ionic liquid. A negligible vapor pressure of [EMIM][B(CN)4] and a sufficient enrichment of Fe(P8+) in a close proximity to the surface enabled XPS measurements as a case study for monitoring direct changes in the electronic structure of the metal centers during redox processes in solution and at liquid/solid interfaces.
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http://dx.doi.org/10.1021/acs.inorgchem.5b00770DOI Listing
July 2015

Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states.

Cell 2014 Aug 7;158(4):778-792. Epub 2014 Aug 7.

Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA; Howard Hughes Medical Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Electronic address:

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.
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http://dx.doi.org/10.1016/j.cell.2014.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263325PMC
August 2014

X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism.

Science 2014 Aug 7;345(6200):1021-6. Epub 2014 Aug 7.

Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Howard Hughes Medical Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.

AMPA-sensitive glutamate receptors are crucial to the structural and dynamic properties of the brain, to the development and function of the central nervous system, and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. We determined multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator, and orthosteric agonists, at 3.8 to 4.1 angstrom resolution. We show how the toxin acts like a straightjacket on the ligand-binding domain (LBD) "gating ring," restraining the domains via both intra- and interdimer cross-links such that agonist-induced closure of the LBD "clamshells" is transduced into an irislike expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in pulling forces on the M3 helices that, in turn, are coupled to ion channel gating.
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http://dx.doi.org/10.1126/science.1258409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263349PMC
August 2014

Measuring cation transport by Na,K- and H,K-ATPase in Xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays.

J Vis Exp 2013 Feb 19(72):e50201. Epub 2013 Feb 19.

Institute of Chemistry, Technical University of Berlin, Germany.

Whereas cation transport by the electrogenic membrane transporter Na(+),K(+)-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H(+),K(+)-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb(+) or Li(+) transport by Na(+),K(+)- or gastric H(+),K(+)-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb(+) (Li(+)) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb(+) uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na(+)/2K(+) transport stoichiometry of the Na(+),K(+)-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H(+),K(+)-ATPase. In principle, the assay is not limited to K(+)-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.
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http://dx.doi.org/10.3791/50201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605618PMC
February 2013

Control of gastric H,K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes.

PLoS One 2012 20;7(3):e33645. Epub 2012 Mar 20.

Institute of Chemistry, Technical University of Berlin, Berlin, Germany.

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E(1)P and E(2)P states and measured Rb(+) uptake under various ionic and pH conditions. The steady-state E(1)P/E(2)P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb(+) uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E(1)P/E(2)P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V(0.5), the voltage, at which the E(1)P/E(2)P ratio is 50∶50, by -100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E(1)P→E(2)P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb(+) uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E(1)P→E(2)P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na(+) profoundly alters the voltage-dependent E(1)P/E(2)P distribution indicating that Na(+) ions can act as surrogates for protons regarding the E(2)P→E(1)P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033645PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308979PMC
August 2012

Volume profile analysis for the reversible binding of superoxide to form iron(II)-superoxo/Iron(III)-peroxo porphyrin complexes.

Inorg Chem 2010 Dec 8;49(23):11254-60. Epub 2010 Nov 8.

Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, Egerlandstrasse 1, 91058 Erlangen, Germany.

The one-electron reduced iron(II)-dioxygen adduct, {Fe(II)-O(2)}(-), is known to be an important intermediate in the catalytic cycle of heme (mono)oxygenases. The same type of species, considered as Fe(III)-peroxo, can be formed in a direct reaction between a Fe(II) center and superoxide. In a unique high-pressure study of the reaction between superoxide and the Fe(II) complex of a crown ether porphyrin conjugate in dimethylsulfoxide (DMSO), the overall Fe(II)-superoxide interaction mechanism could be visualized and the nature of all species that occur along the reaction coordinate could be clarified. The equilibrium between the low-spin and high-spin forms of the starting Fe(II) complex was quantified, which turns out to be the actual activation step toward substitution and subsequent inner-sphere electron transfer reactions. The constructed reaction volume profile demonstrates that the reaction product consists of Fe(III)-peroxo and Fe(II)-superoxo species that exist in equilibrium, which can better account for the versatile reactivity of {Fe(II)-O(2)}(-) adducts toward different substrates.
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http://dx.doi.org/10.1021/ic102092hDOI Listing
December 2010

Deceleration of the E1P-E2P transition and ion transport by mutation of potentially salt bridge-forming residues Lys-791 and Glu-820 in gastric H+/K+-ATPase.

J Biol Chem 2010 Dec 4;285(50):39366-79. Epub 2010 Oct 4.

Technical University of Berlin, Institute of Chemistry, Secr. PC 14, Strasse des 17. Juni 135, D-10623 Berlin, Germany.

A lysine residue within the highly conserved center of the fifth transmembrane segment in P(IIC)-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK(a) modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K(+)-induced stationary currents. In Rb(+) uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E(2)-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E(1)P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E(1)P ↔ E(2)P conformational transition, thus explaining the E(1)P shift and the reduced Rb(+) transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb(+) transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E(2)P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H(+) pumping P-type ATPases.
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http://dx.doi.org/10.1074/jbc.M110.133470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998106PMC
December 2010

Effects of frozen conditions on stereoselectivity and velocity of O-glycosylation reactions.

Bioorg Med Chem 2010 Jun 9;18(11):3687-95. Epub 2010 Apr 9.

RIKEN.Advanced Science Institute, Wako, Saitama 351-0198, Japan.

Rate acceleration of O-glycosylation had been observed in p-xylene under frozen conditions, when thioglycosides were activated by methyl trifluoromethane sulfonate. Curiously, significant perturbation of stereoselectivity was observed. Effects of various factors, such as solvent, concentration, anomeric configuration and protective groups of the donor, were systematically examined to clarify the mechanistic implications of stereoselectivity on glycosylation under frozen system. Our study revealed that the stereoselectivity was affected by concentration both in liquid as well as in frozen conditions, indicating that rate acceleration effect in frozen solvent was caused by highly concentrated environments.
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http://dx.doi.org/10.1016/j.bmc.2010.04.013DOI Listing
June 2010

Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

J Gen Physiol 2010 Feb;135(2):115-34

Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany.

The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.
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http://dx.doi.org/10.1085/jgp.200910301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812498PMC
February 2010

Chelate electronic properties control the redox behaviour and superoxide reactivity of seven-coordinate manganese(II) complexes.

Dalton Trans 2009 Aug 23(32):6292-5. Epub 2009 Jun 23.

Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, Egerlandstr. 1, 91058, Erlangen, Germany.

We have synthesized and characterized two Mn(II) seven-coordinate complexes with N5 pentadentate ligands, which contain hydrazone and hydrazide groups respectively. We have shown that insertion of hydrazido (amido) groups into the ligand sphere increases the negative charge of the chelate, without changing a donor atom set and coordination geometry, and radically modulate a redox activity of seven-coordinate manganese complexes, which is important for the function of manganese as a superoxide dismutase catalytic center.
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http://dx.doi.org/10.1039/b906100mDOI Listing
August 2009

E2P state stabilization by the N-terminal tail of the H,K-ATPase beta-subunit is critical for efficient proton pumping under in vivo conditions.

J Biol Chem 2009 Jul 2;284(30):20147-54. Epub 2009 Jun 2.

Institute of Chemistry, Technical University of Berlin, D-10623 Berlin, Germany.

The catalytic alpha-subunits of Na,K- and H,K-ATPase require an accessory beta-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the beta-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated beta-variants were expressed in Xenopus oocytes, together with the S806C alpha-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E(1)P/E(2)P states. Whereas the E(1)P/E(2)P conformational equilibrium was unaffected for the shorter N-terminal deletions betaDelta4 and betaDelta8, we observed significant shifts toward E(1)P for the two larger deletions betaDelta13 and betaDelta29. Moreover, the reduced DeltaF/F ratios of betaDelta13 and betaDelta29 indicated an increased reverse reaction via E(2)P --> E(1)P + ADP --> E(1) + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E(2)P-specific inhibitor SCH28080, which becomes especially apparent at high concentrations (100 microm). Despite unaltered apparent Rb(+) affinities, the maximal Rb(+) uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the beta-N terminus and alpha-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase beta-subunit may stabilize the pump in the E(2)P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the beta-N terminus of the closely related Na,K-ATPase does not affect the E(1)P/E(2)P distribution or pump activity, indicating that the E(2)P-stabilizing effect by the beta-N terminus is apparently a unique property of the H,K-ATPase.
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http://dx.doi.org/10.1074/jbc.M109.005769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740441PMC
July 2009

Impaired plasma membrane targeting or protein stability by certain ATP1A2 mutations identified in sporadic or familial hemiplegic migraine.

Channels (Austin) 2009 Mar-Apr;3(2):82-7. Epub 2009 Mar 3.

Technical University of Berlin, Institute of Chemistry, Berlin, Germany.

Mutations in three different genes have been implicated in familial hemiplegic migraine (FHM), two of them code for neuronal voltage-gated cation channels, CACNA1A and SCN1A, while the third encodes ATP1A2, the alpha(2)-isoform of the Na(+)/K(+)-ATPase's catalytic subunit, thus classifying FHM as an ion channel/ion transporter disorder. The Na(+)/K(+)-ATPase maintains the physiological gradients for Na(+) and K(+) ions and is therefore critical for the activity of ion channels and transporters involved in neurotransmitter uptake or Ca(2+) signaling. Diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations, which reach far beyond simple loss-of-function. We have shown recently that ATP1A2 mutations frequently lead to changes in the enzyme's voltage-dependent properties, kinetics or apparent cation affinities. Here, we present functional data on a so far uncharacterized set of ATP1A2 mutations (G301R, R908Q and P979L) upon expression in Xenopus oocytes and HEK293FT cells, and provide evidence for a novel pathophysiological mechanism. Whereas the G301R mutant was inactive, no functional changes were observed for mutants R908Q and P979L in the oocyte expression system. However, the R908Q mutant was less effectively expressed in the plasma membrane of oocytes, making it the first missense mutation to result in defective plasma membrane targeting. Notably, the P979L mutant exhibited the same cellular expression profile as the wild-type protein, both in Xenopus oocytes and in transfected HEK293FT cells grown at 28 degrees C, but much less P979L protein was found upon cell growth at 37 degrees C, showing for the first time that temperature-sensitive effects on protein stability can underlie ATP1A2 loss-of-function.
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http://dx.doi.org/10.4161/chan.3.2.8085DOI Listing
September 2009

Functional significance of E2 state stabilization by specific alpha/beta-subunit interactions of Na,K- and H,K-ATPase.

J Biol Chem 2009 Feb 8;284(6):3842-54. Epub 2008 Dec 8.

Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany.

The beta-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic alpha-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K- and gastric H,K-ATPase beta-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K+ that were moderate for the Na,K-ATPase beta-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase beta-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase beta-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na+ competition for extracellular K+ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E2P-E1P conversion was accelerated by the H,K-ATPase beta-(Y44W,Y48W) mutation, and H+ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K- and H,K-ATPase alpha-subunits, which are at interacting distance to the beta-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of alpha- and beta-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions.
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http://dx.doi.org/10.1074/jbc.M808101200DOI Listing
February 2009

A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate.

J Mol Biol 2008 Dec 9;384(4):865-77. Epub 2008 Oct 9.

Department of Biochemistry, Molecular Genetics Group, Johann Wolfgang Goethe University, Frankfurt/Main, Germany.

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.
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http://dx.doi.org/10.1016/j.jmb.2008.09.074DOI Listing
December 2008

Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2.

J Biol Chem 2008 Nov 26;283(45):31097-106. Epub 2008 Aug 26.

Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany.

Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic hemiplegic migraine, including missense mutations (T263M, T376M, R383H, A606T, R763H, M829R, R834Q, R937P, and X1021R), a deletion mutant (del(K935-S940)ins(I)), and a frameshift mutation (S966fs). According to the Na(+)/K(+)-ATPase crystal structure, a subset of the mutated residues (Ala(606), Arg(763), Met(829), and Arg(834)) is involved in important interdomain H-bond networks, and the C terminus of the enzyme, which is elongated by the X1021R mutation, has been implicated in voltage dependence and formation of a third Na(+)-binding site. Upon heterologous expression in Xenopus oocytes, the analysis of electrogenic transport properties, Rb(+) uptake, and protein expression revealed pronounced and markedly diverse functional alterations in all ATP1A2 mutants. Abnormalities included a complete loss of function (T376M), impaired plasma membrane expression (del(K935-S940)ins(I) and S966fs), and altered apparent affinities for extracellular cations or reduced enzyme turnover (R383H, A606T, R763H, R834Q, and X1021R). In addition, changes in the voltage dependence of pump currents and the increased rate constants of the voltage jump-induced redistribution between E(1)P and E(2)P states were observed. Thus, mutations that disrupt distinct interdomain H-bond patterns can cause abnormal conformational flexibility and exert long range consequences on apparent cation affinities or voltage dependence. Of interest, the X1021R mutation severely impaired voltage dependence and kinetics of Na(+)-translocating partial reactions, corroborating the critical role of the C terminus of Na(+)/K(+)-ATPase in these processes.
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http://dx.doi.org/10.1074/jbc.M802771200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662176PMC
November 2008

Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes.

Biochemistry 2008 Apr 15;47(14):4288-97. Epub 2008 Mar 15.

Max Volmer Laboratory for Biophysical Chemistry, Institute of Chemistry, Technical University of Berlin, Secr. PC 14, Strasse des 17. Juni 135, D-10623 Berlin, Germany.

The role of N-linked glycosylation of beta-subunits in the functional properties of the oligomeric P-type ATPases Na,K- and H,K-ATPase has been examined by expressing glycosylation-deficient Asn-to-Gln beta-variants in Xenopus oocytes. For both ATPases, the absence of the huge N-linked oligosaccharide moiety on the beta-subunit does not affect alpha/beta coassembly, plasma membrane delivery or functional activity of the holoenzyme. Whereas this is in line with several previous glycosylation studies on Na,K-ATPase, this is the first report showing that the cell surface delivery and enzymatic activity of the gastric H,K-ATPase is unaffected by the lack of N-linked glycosylation. Sulfhydryl-specific labeling of introduced cysteine reporter sites with the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes enabled us to further investigate potential effects of the N-glycans on more subtle enzymatic properties, like the distribution between E 1P/E 2P states of the catalytic cycle and the kinetics of the E 1P/E 2P conformational transition under presteady state conditions. For both Na,K-ATPase and H,K-ATPase, we observed differences in neither the voltage-dependent E 1P/E 2P ratio nor the kinetics of the E 1P/E 2P transition between holoenzymes comprising glycosylated and glycosylation-deficient beta-subunits. We conclude that the N-linked glycans on these essential accessory subunits of oligomeric P-type ATPases are dispensable for proper folding, membrane stabilization of the alpha-subunit and transport function itself. Glycosylation is rather important for other cellular functions not relevant in the oocyte expression system, such as intercellular interactions or basolateral versus apical targeting in polarized cells, as demonstrated in other expression systems.
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http://dx.doi.org/10.1021/bi800092kDOI Listing
April 2008

Iron(III) complex of a crown ether-porphyrin conjugate and reversible binding of superoxide to its Iron(II) form.

J Am Chem Soc 2007 Apr 20;129(14):4217-28. Epub 2007 Mar 20.

Institute of Inorganic Chemistry, University of Erlangen-Nürnberg, Egerlandstrasse 1, 91058 Erlangen, Germany.

The synthesis and characterization of the Fe(III) complex of a novel crown ether-porphyrin conjugate, 52-N-(4-aza-18-crown-6)methyl-54,104,154,204-tetra-tert-butyl-56-methyl-5,10,15,20-tetraphenylporphyrin (H2Porph), as well as the corresponding hydroxo, dimeric, Fe(II), and peroxo species are reported. The crystal structure of [FeIII(Porph)Cl].H3O+.FeCl4-.C6H6.EtOH is also reported. [FeIII(Porph)(DMSO)2]+ and K[FeIII(Porph)(O22-)] are high-spin species (Mössbauer data: delta = 0.38 mm s(-1), DeltaEq = 0.83 mm s(-1) and delta = 0.41 mm s(-1), DeltaEq = 0.51 mm s(-1), respectively), whereas in a solution of reduced [FeIII(Porph)(DMSO)2]+ complex the low-spin [FeII(Porph)(DMSO)2] (delta = 0.44 mm s(-1), DeltaEq = 1.32 mm s(-1)) and high-spin [FeII(Porph)(DMSO)] (delta = 1.27 mm s(-1), DeltaEq = 3.13 mm s(-1)) iron(II) species are observed. The reaction of [FeIII(Porph)(DMSO)2]+ with KO2 in DMSO has been investigated. The first reaction step, involving reduction to [FeII(Porph)(DMSO)2], was not investigated in detail because of parallel formation of an Fe(III)-hydroxo species. The kinetics and thermodynamics of the second reaction step, reversible binding of superoxide to the Fe(II) complex and formation of an Fe(III)-peroxo species, were studied in detail (by stopped-flow time-resolved UV/vis measurements in DMSO at 25 degrees C), resulting in kon = 36 500 +/- 500 M(-1) s(-1), koff = 0.21 +/- 0.01 s(-1) (direct measurements using an acid as a superoxide scavenger), and KO2- = (1.7 +/- 0.2) x 10(5) (superoxide binding constant kinetically obtained as kon/koff), (1.4 +/- 0.1) x 10(5), and (9.0 +/- 0.1) x 10(4) M(-1) (thermodynamically obtained in the absence and in the presence of 0.1 M NBu4PF6, respectively). Temperature-dependent kinetic measurements for kon (-40 to 25 degrees C in 3:7 DMSO/CH3CN mixture) yielded the activation parameters DeltaH = 61.2 +/- 0.9 kJ mol(-1) and DeltaS = +48 +/- 3 J K(-1) mol(-1). The observed reversible binding of superoxide to the metal center and the obtained kinetic and thermodynamic parameters are unique. The finding that fine-tuning of the proton concentration can cause the Fe(III)-peroxo species to release O2- and form an Fe(II) species is of biological interest, since this process might occur under very specific physiological conditions.
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http://dx.doi.org/10.1021/ja064984pDOI Listing
April 2007

Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidase.

FEBS J 2005 Jan;272(2):404-12

Institut für Biochemie, Abteilung Molekulare Genetik, Johann Wolfgang Goethe-Universität, Frankfurt-am-Main, Germany.

In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78(II)) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78(II)Q) or reduce it to about 50% (E78(II)A); in the latter case, the mutation causes no drastic decrease in heme a(3) reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78(II) is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at approximately 1760 to 1710 cm(-1) excludes the protonation of E78(II) in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm(-1) reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78(II) in the Paracoccus enzyme.
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http://dx.doi.org/10.1111/j.1742-4658.2004.04480.xDOI Listing
January 2005