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Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Strukturbiokemi)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Strukturbiokemi)
2007 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 448, no 7153, 613-616 p.Article in journal (Refereed) Published
Abstract [en]

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the omega-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

Place, publisher, year, edition, pages
2007. Vol. 448, no 7153, 613-616 p.
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-27058DOI: 10.1038/nature06009ISI: 000248446700048OAI: oai:DiVA.org:su-27058DiVA: diva2:212541
Note
Totalt antal författare: 10Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Membrane Protein Tailoring and Structural Studies of Leukotriene C4 Synthase
Open this publication in new window or tab >>Membrane Protein Tailoring and Structural Studies of Leukotriene C4 Synthase
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Despite a dramatic increase in the number of proteins that have been structurally characterized in recent years, there are still less than 200 unique structures of membrane proteins known today. This is only 1% of the total number of unique protein structures found in structural databases worldwide. There are several reasons for this hindered progress in the structure determination of membrane proteins; it is difficult to generate membrane proteins in recombinant expression systems and it requires the use of appropriate detergents, both for membrane extraction and to keep them stable in solution. This makes isolation and purification problematic. Importantly, once isolated, these proteins are notoriously difficult to crystallize for X-ray structure determination.

 In this thesis, I present two techniques that can be used to increase the likelihood of success in the structural determination of membrane proteins. I started by focusing on problems that occur at an early stage of the process, where I developed a directed-evolution method to overcome problems with low yields during membrane protein production. In addition, I describe a screen for optimal detergent usage when purifying and crystallizing recombinant membrane proteins in eukaryotic hosts.

 The crystal structure of human Leukotriene C4 synthase has been solved. This is the first human membrane protein whose structure has been solved at high resolution. The model provides a structural basis for the formation of potent lipid mediators, which are implicated in the pathophysiology of asthma and chronic inflammation. Furthermore, the structure reveals insight into how specificity can be achieved for lipophilic substrate molecules. In addition, I have determined the crystal structure of a hexahistidine tag and use it to describe the molecular basis of the single most used chromatography method, IMAC.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 68 p.
Keyword
membrane proteins, random mutagenesis, detergent screening, Leukotriene C₄ synthase, IMAC
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-27067 (URN)978-91-7155-882-4 (ISBN)
Public defence
2009-05-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A , Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2009-05-06 Created: 2009-04-22 Last updated: 2009-04-24Bibliographically approved
2. Structural biology of integral membrane proteins - From methods to molecular mechanisms
Open this publication in new window or tab >>Structural biology of integral membrane proteins - From methods to molecular mechanisms
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure.  Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis  are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials.

Despite the challenges in membrane protein structure determination two structures are presented in the thesis:

The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway.

The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy, 2009. 59 p.
Keyword
membrane proteins, CorA, magnesium transport, screening, Leukotriene C4 synthase, detergents
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-30069 (URN)978-91-628-7899-3 (ISBN)
Public defence
2009-10-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2009-10-07 Created: 2009-10-01 Last updated: 2012-08-10Bibliographically approved

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