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Structure and lipid interactions of membrane-associated glycosyltransferases: Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Lena Mäler)ORCID iD: 0000-0003-2965-2873
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 [en]
glycosyltransferase, monotopic membrane proteins, galactolipids, NMR, chitin synthase, DGD2, LPS, WaaG, MIT domain
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-131084ISBN: 978-91-7649-435-6OAI: oai:DiVA.org:su-131084DiVA: diva2:936254
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
List of papers
1. Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana
Open this publication in new window or tab >>Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana
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2011 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 21, 4451-4466 p.Article in journal (Refereed) Published
Abstract [en]

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227–245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130–148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227–245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62012 (URN)10.1021/bi200162f (DOI)000290837400008 ()
Funder
Swedish Research Council, 621-2011-5964
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2016-06-15
2. Insights into the membrane interacting properties of the C-terminal domain of the monotopic glycosyltransferase DGD2 in A. thaliana
Open this publication in new window or tab >>Insights into the membrane interacting properties of the C-terminal domain of the monotopic glycosyltransferase DGD2 in A. thaliana
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-131248 (URN)
Available from: 2016-06-14 Created: 2016-06-14 Last updated: 2016-06-15Bibliographically approved
3. Membrane Interaction of the Glycosyltransferase WaaG
Open this publication in new window or tab >>Membrane Interaction of the Glycosyltransferase WaaG
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.

National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-120191 (URN)10.1016/j.bpj.2015.06.036 (DOI)000359180400012 ()
Available from: 2015-09-04 Created: 2015-09-02 Last updated: 2016-06-15Bibliographically approved
4. Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases
Open this publication in new window or tab >>Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases
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2016 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 16, 3072-3088 p.Article in journal (Refereed) Published
Abstract [en]

Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a Microtubule Interacting and Trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed and characterized. Both were shown to interact strongly with the plasma membrane component phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modelling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes. 

Keyword
chitin synthase, microtubule interacting and trafficking domain, NMR structure, oomycete, phospholipids
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-131279 (URN)10.1111/febs.13794 (DOI)
Available from: 2016-06-15 Created: 2016-06-15 Last updated: 2016-09-01Bibliographically approved

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