Susanna Törnroth-Horsefield

University of Gothenburg, Göteborg, Vaestra Goetaland, Sweden

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Publications (13)111.07 Total impact

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    ABSTRACT: Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.
    Proceedings of the National Academy of Sciences 04/2014; · 9.81 Impact Factor
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    ABSTRACT: Water transport across cellular membranes is mediated by a family of membrane proteins known as aquaporins (AQP). AQPs were first discovered based on their ability to be inhibited by mercurial compounds, an experiment which has followed the aquaporin field ever since. While mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane aquaporins are known to be gated by phosphorylation, pH and Ca2+. We have previously solved the structure of the spinach AQP SoPIP2;1 in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2;1 we solved the structure of the SoPIP2;1-mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteines. Contrary to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2;1, an effect which could not be attributed to any of the cysteines. This indicates that other factors might influence the effect of mercury on SoPIP2;1, one of which could be the properties of the lipid bilayer.
    Biochemical Journal 07/2013; · 4.65 Impact Factor
  • Anna Frick, Michael Järvå, Susanna Törnroth-Horsefield
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    ABSTRACT: Plants have evolved to cope with fluctuations in water supply by gating their water channels known as aquaporins. During flooding, a rapid drop of cytosolic pH due to anoxia leads to a simultaneous closure of the aquaporins in the plasma membrane. The closing mechanism has been suggested to involve a conserved histidine on cytosolic loop D. Here we report the crystal structure of a spinach aquaporin at low pH, revealing for the first time the structural basis for how this pH-sensitive histidine helps to keep the aquaporin in a closed state. Structured summary of protein interactions: SoPIP2;1andSoPIP2;1bindbyx-ray crystallography(View interaction).
    FEBS letters 02/2013; · 3.54 Impact Factor
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    Alaa Adawy, Susanna Törnroth-Horsefield, Willem de Grip, Willem van Enckevort, Elias Vlieg
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    ABSTRACT: The growth of high quality single protein crystals, yielding the highest X-ray resolution, remains a bottleneck in the macromolecular crystallography. Here we show an entirely convection-free crystallization environment, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the “ceiling” of a growth-cell completely filled with the crystallization solution. The “ceiling crystals” experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on a number of proteins and, the ceiling crystals diffracted X-rays to resolution limits beyond their current world records, even while commercial preparations of sub-optimal purity were used. This also demonstrates the intrinsic purification effect of the ceiling crystallization method.
    14 th International Conference on the Crystallization of Biological Macromolecules; 09/2012
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    Alaa Adawy, Susanna Törnroth-Horsefield, Willem de Grip, Willem van Enckevort, Elias Vlieg
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    ABSTRACT: Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional functional structure of biological macromolecules. The growth of high quality single crystals, yielding the highest X-ray resolution, remains a bottleneck in this methodology. Gravity-driven convection is hold responsible for interfering with the growth of perfect crystals in all terrestrial methods, while space microgravity provides diffusive mass transport which can improve the quality of protein crystals. Here we show that through an efficient modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. In this novel method, dubbed “Ceiling Crystallization Method”, we exploit gravity to achieve diffusion-limited crystal growth in a terrestrial setting instead of eliminating it. Protein crystals grow at the “ceiling” of a growth-cell experience the same effects as in space microgravity experiments. The development of the depletion zone around the ceiling crystals was monitored by using phase shift interferometery. The development of a concentration gradient, is assisted by the action of gravity, insuring that the mass transport in this case is diffusion limited. The new method was tested on different proteins and in all cases, ceiling crystals diffracted X-rays to resolution limits beyond their current world records, even while commercial preparations of sub-optimal purity were used. This demonstrates the enhanced purification effect of the ceiling crystallization method.
    4th European Conference on Crystal Growth (ECCG4); 06/2012
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    ABSTRACT: Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions.
    FEBS letters 04/2010; 584(12):2580-8. · 3.54 Impact Factor
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    ABSTRACT: Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca(2+) can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C(alpha) atom of Glu31 10 A away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.
    Journal of Molecular Biology 03/2009; 387(3):653-68. · 3.91 Impact Factor
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    Susanna Törnroth-Horsefield, Richard Neutze
    Proceedings of the National Academy of Sciences 01/2009; 105(50):19565-6. · 9.81 Impact Factor
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    ABSTRACT: This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
    Acta Crystallographica Section D Biological Crystallography 12/2008; 64(Pt 11):1183-6. · 12.67 Impact Factor
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    ABSTRACT: Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-A resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.
    Proceedings of the National Academy of Sciences 10/2008; 105(36):13327-32. · 9.81 Impact Factor
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    ABSTRACT: Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.
    Structure 01/2008; 15(12):1663-73. · 5.99 Impact Factor
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    ABSTRACT: An acceleration in the rate at which new aquaporin structures are determined means that structural models are now available for mammalian AQP0, AQP1, AQP2 and AQP4, bacterial GlpF, AqpM and AQPZ, and the plant SoPIP2;1. With an apparent consensus emerging concerning the mechanism of selective water transport and proton extrusion, emphasis has shifted towards the issues of substrate selectivity and the mechanisms of aquaporin regulation. In particular, recently determined structures of plant SoPIP2;1, sheep and bovine AQP0, and Escherichia coli AQPZ provide new insights into the underlying structural mechanisms by which water transport rates are regulated in diverse organisms. From these results, two distinct pictures of 'capping' and 'pinching' have emerged to describe aquaporin gating.
    Current Opinion in Structural Biology 09/2006; 16(4):447-56. · 8.74 Impact Factor
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    ABSTRACT: Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Channel closure results either from the dephosphorylation of two conserved serine residues under conditions of drought stress, or from the protonation of a conserved histidine residue following a drop in cytoplasmic pH due to anoxia during flooding. Here we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 A resolution and in its open conformation at 3.9 A resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation loop D is displaced up to 16 A and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins.
    Nature 03/2006; 439(7077):688-94. · 38.60 Impact Factor

Publication Stats

511 Citations
111.07 Total Impact Points

Institutions

  • 2008–2013
    • University of Gothenburg
      • • Institutionen för kemi och molekylärbiologi
      • • Department of Cell- and Molecular Biology
      Göteborg, Vaestra Goetaland, Sweden
  • 2006–2009
    • Chalmers University of Technology
      • Department of Chemical and Biological Engineering
      Göteborg, Vaestra Goetaland, Sweden