title: The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA-binding site.
abstract: Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants.
authors.affiliation: Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Maes
authors.affiliation: Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Einav
authors.initials: E
authors.lastname: Amit
authors.affiliation: Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Tsafi
authors.initials: T
authors.lastname: Danieli
authors.affiliation: Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Mario
authors.initials: M
authors.lastname: Lebendiker
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Abraham
authors.initials: A
authors.lastname: Loyter
authors.affiliation: Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel assaf.friedler@mail.huji.ac.il.
authors.forename: Assaf
authors.initials: A
authors.lastname: Friedler
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D007473:Ion Channels; D011994:Recombinant Proteins; C432104:VIP1 protein, Arabidopsis; C051583:virE2 protein, Agrobacterium
keywords: Agrobacterium; VIP1; VirE2; peptide arrays; protein expression and purification