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Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, 2725-2735 p.Article in journal (Refereed) Published
Abstract [en]

The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

Place, publisher, year, edition, pages
2014. Vol. 42, no 4, 2725-2735 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-102485DOI: 10.1093/nar/gkt1119ISI: 000332381000059OAI: oai:DiVA.org:su-102485DiVA: diva2:710505
Funder
Swedish Research Council, 2010-5200The Wenner-Gren FoundationSwedish Foundation for Strategic Research Carl Tryggers foundation EU, FP7, Seventh Framework Programme
Note

AuthorCount:9;

Available from: 2014-04-07 Created: 2014-04-07 Last updated: 2017-12-05Bibliographically approved
In thesis
1. 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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Berntsson, Ronnie P. -A.Odegrip, RichardSehlén, WilhelminaSkaar, KarinHögbom, MartinStenmark, Pål
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