title: Interaction studies of the human and Arabidopsis thaliana Med25-ACID proteins with the herpes simplex virus VP16- and plant-specific Dreb2a transcription factors.
abstract: Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional co-regulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.
authors.affiliation: Department of Chemistry, Umeå University, Umeå, Sweden.
authors.forename: Ximena
authors.initials: X
authors.lastname: Aguilar
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Blomberg
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Brännström
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Olofsson
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schleucher
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Björklund
mesh_terms: D017360:Arabidopsis / Q000378:metabolism* / Q000821:virology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D056892:Mediator Complex / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C114579:DREB2A protein, Arabidopsis; D017405:Herpes Simplex Virus Protein Vmw65; C483045:MED25 protein, human; D056892:Mediator Complex; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C475686:PFT1 protein, Arabidopsis; D014157:Transcription Factors