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Membrane Interaction of the Glycosyltransferase WaaG
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Number of Authors: 4
2015 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 109, no 3, 552-563 p.Article in journal (Refereed) Published
Abstract [en]

The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely a-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane.

Place, publisher, year, edition, pages
2015. Vol. 109, no 3, 552-563 p.
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-120191DOI: 10.1016/j.bpj.2015.06.036ISI: 000359180400012OAI: oai:DiVA.org:su-120191DiVA: diva2:851334
Available from: 2015-09-04 Created: 2015-09-02 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Structure and lipid interactions of membrane-associated glycosyltransferases: Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes
Open this publication in new window or tab >>Structure and lipid interactions of membrane-associated glycosyltransferases: Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway.

From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2016. 81 p.
Keyword
glycosyltransferase, monotopic membrane proteins, galactolipids, NMR, chitin synthase, DGD2, LPS, WaaG, MIT domain
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-131084 (URN)978-91-7649-435-6 (ISBN)
External cooperation:
Public defence
2016-09-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2016-08-24 Created: 2016-06-13 Last updated: 2016-09-01Bibliographically approved
2. Taming the Griffin: Membrane interactions of peripheral and monotopic glycosyltransferases and dynamics of bacterial and plant lipids in bicelles
Open this publication in new window or tab >>Taming the Griffin: Membrane interactions of peripheral and monotopic glycosyltransferases and dynamics of bacterial and plant lipids in bicelles
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria.

To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle.

In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms.

WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. 77 p.
Keyword
membrane, bicelle, lipid, detergent, lipopolysaccharide, glycosyltransferase, WaaG, fluorescence, circular dichroism, NMR, paramagnetic relaxation enhancement, model-free approach, dynamics
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-146872 (URN)978-91-7649-978-8 (ISBN)978-91-7649-979-5 (ISBN)
Public defence
2017-11-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.

Available from: 2017-10-11 Created: 2017-09-14 Last updated: 2017-10-05Bibliographically approved

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