Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana
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.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2011. Vol. 50, no 21, 4451-4466 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-62012DOI: 10.1021/bi200162fISI: 000290837400008OAI: oai:DiVA.org:su-62012DiVA: diva2:439386
Funder
Swedish Research Council, 621-2011-5964
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2017-12-08
In thesis
1. Property-controlling Enzymes at the Membrane Interface
Open this publication in new window or tab >>Property-controlling Enzymes at the Membrane Interface
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Monotopic proteins represent a specialized group of membrane proteins in that they are engaged in biochemical events taking place at the membrane interface. In particular, the monotopic lipid-synthesizing enzymes are able to synthesize amphiphilic lipid products by catalyzing two biochemically distinct molecules (substrates) at the membrane interface. Thus, from an evolutionary point of view, anchoring into the membrane interface enables monotopic enzymes to confer sensitivity to a changing environment by regulating their activities in the lipid biosynthetic pathways in order to maintain a certain membrane homeostasis. We are focused on a plant lipid-synthesizing enzyme DGD2 involved in phosphate shortage stress, and analyzed the potentially important lipid anchoring segments of it, by a set of biochemical and biophysical approaches. A mechanism was proposed to explain how DGD2 adjusts its activity to maintain a proper membrane. In addition, a multivariate-based bioinformatics approach was used to predict the lipid-binding segments for GT-B fold monotopic enzymes. In contrast, a soluble protein Myr1 from yeast, implicated in vesicular traffic, was also proposed to be a membrane stress sensor as it is able to exert different binding properties to stressed membranes, which is probably due to the presence of strongly plus-charged clusters in the protein. Moreover, a bacterial monotopic enzyme MGS was found to be able to induce massive amounts of intracellular vesicles in Escherichia coli cells. The mechanisms involve several steps: binding, bilayer lateral expansion, stimulation of lipid synthesis, and membrane bending. Proteolytic and mutant studies indicate that plus-charged residues and the scaffold-like structure of MGS are crucial for the vesiculation process. Hence, a number of features are involved governing the behaviour of monotopic membrane proteins at the lipid bilayer interface.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 80 p.
Keyword
monotopic membrane protein, lipid-protein interaction, membrane curvature, glycosyltransferase, Rossmann fold
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-61988 (URN)978-91-7447-330-8 (ISBN)
Public defence
2011-10-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.Available from: 2011-09-29 Created: 2011-09-06 Last updated: 2011-11-18Bibliographically approved
2. 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

Open Access in DiVA

Fulltext(10060 kB)327 downloads
File information
File name FULLTEXT04.pdfFile size 10060 kBChecksum SHA-512
af777eed8bfaeb0eff488fca5b17a039066f61d12c3bd2b7079880c7550f989cffa853ae45ddd40307f41e12bbc402dca8bc7ceadd031e7ee69495bb85e73c66
Type fulltextMimetype application/pdf

Other links

Publisher's full text

Search in DiVA

By author/editor
Szpryngiel, ScarlettGe, ChangrongLind, JesperWieslander, ÅkeMäler, Lena
By organisation
Department of Biochemistry and Biophysics
In the same journal
Biochemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 327 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 160 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf