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Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats
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 Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, 7778-7790 p.Article in journal (Refereed) Published
Abstract [en]

As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.

Place, publisher, year, edition, pages
2010. Vol. 38, no 21, 7778-7790 p.
Keyword [en]
DNA-binding protein, direct repeats, P2 C repressor
National Category
Structural Biology
Research subject
Structural Biology; Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-42003DOI: 10.1093/nar/gkq626ISI: 000284952000042OAI: oai:DiVA.org:su-42003DiVA: diva2:343481
Funder
The Wenner-Gren FoundationSwedish Foundation for Strategic Research Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Structural Studies of Flexible Biomolecules and a DNA-binding Protein
Open this publication in new window or tab >>Structural Studies of Flexible Biomolecules and a DNA-binding Protein
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 76 p.
Keyword
Maximum entropy, motilin, DNA-binding proteins, Karplus equation, disaccaride, direct repeats
National Category
Structural Biology
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-42009 (URN)978-91-7447-102-1 (ISBN)
Public defence
2010-09-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 09:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript. Available from: 2010-08-23 Created: 2010-08-13 Last updated: 2010-08-17Bibliographically approved
2. A Genetic Switch in Bacteriophages within the Peduovirinae Subfamily: Structure, Function and Evolution
Open this publication in new window or tab >>A Genetic Switch in Bacteriophages within the Peduovirinae Subfamily: Structure, Function and Evolution
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The temperate bacteriophages in the Peduovirinae subfamily can either grow lytically or integrate into their bacterial host and form lysogeny. Which one of the two life cycles the phage will enter after infection is controlled by a transcriptional switch. The switch also controls the induction of genes necessary for an integrated phage, a prophage, to excise out of the host genome and propagate lytically. In its most simple form, the transcriptional switch consists of two proteins repressing each other’s promoters, which are oriented face to face in close proximity.

The Peduovirinae phages contain two types of transcriptional switches. They were studied with phylogenetic methods to determine their evolution and distribution. Bioinformatic analyses showed that there were several new E. coli integration sites and new inferred immunity classes among the Peduovirinae phages. The two switch types fell into two distinct groups, with no overlap in any of the proteins, but these groups were not defined by host barriers. But in vivo distribution did show a host preference.

The P2 C protein was crystallized and its 3D structure determined. It forms a symmetrical dimer in vitro, with an unstructured C-terminal end. The DNA binding domain was determined to lie in alpha helix three and narrowed down to three residues. The C terminal end of the protein is suggested to be part of tetramerization, but a nine amino acid truncation does not affect activity in vitro.

In an attempt to discover the mechanism between the switch from lysogeny to lysis in phage P2 the interactions between the two switch proteins and the proteins of its host E. coli was analyzed. Eight E. coli proteins interacted with protein C or Cox, but no interaction between the two switch proteins was detected. Two E. coli proteins showed a distinct effect on the expression of C, and several affected the level of phage lysis. The mechanisms behind these effects are still unclear.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2012. 88 p.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-74031 (URN)978-91-7447-467-1 (ISBN)
Public defence
2012-03-30, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr 3: ManuscriptAvailable from: 2012-03-08 Created: 2012-02-27 Last updated: 2012-03-01Bibliographically approved
3. Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins
Open this publication in new window or tab >>Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biochemical studies of proteins are crucial for a more detailed view of the world around us. The focus of biochemical studies can vary, from a complex mammalian system to a more simple viral entity, but the same methods and principles apply. In biochemistry one rely on both in vitro and in vivo analyses to understand biological processes. Protein crystallography has since the late 1950s been an additional important tool. By visualizing the structures of molecules involved in a biological process one can truly comprehend the molecular mechanisms of an organism or cell at the chemical level. This thesis includes structural biochemical work in combination with mutational and functional studies of proteins from both human and virus.

Human tetraspanins are integral membrane proteins grouped by their conserved structural features. Many of them have been shown to regulate cell migration, fusion, and signalling in the cell by functioning as organizers of multi-molecular membrane complexes. Several tetraspanins are also implicated in different forms of human cancers. How tetraspanins perform their function is still not known at the molecular level and today very little structural data exist on complete tetraspanin proteins. Structural biochemical studies require mg quantities of purified protein, something that is not easily obtained for membrane proteins. This thesis includes a family-wide approach to achieve full-length tetraspanins for biochemical studies. To facilitate this process a GFP-based optimization scheme for production and purification of membrane proteins in E. coli and S. cerevisiae has been applied. By utilizing this approach, we identified 8 human tetraspanins that can be produced and isolated from either E. coli or S. cerevisiae, and in one case using either system.

The temperate bacteriophage P2 is a virus, which can enter both the lytic and the lysogenic cycle upon infection of its host. The outcome of the infection is regulated by and dependent on several proteins encoded by the viral genome. The immunity repressor P2 and the Cox repressor direct the phage into either cycle. Integration and excision of the virus DNA requires the enzyme P2 integrase. The work in this thesis presents high-resolution crystal structures of these key proteins from the regulation of lysogeny in bacteriophage P2. By using a crystallographic approach in combination with mutational studies, key characteristics of these three proteins are presented. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 52 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-119225 (URN)978-91-7649-209-3 (ISBN)
Public defence
2015-09-18, 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 3: Manuscript.

Available from: 2015-08-27 Created: 2015-08-03 Last updated: 2015-08-20Bibliographically approved

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