Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors

• Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator 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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Keyword

Mediator

Med25

Dreb2a

VP16

conformational changes

NMR

ITC

Publication and Content Type

ref (subject category)

art (subject category)