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.

Figure 1. A.thaliana and human Med25-ACIDs interacts with the Herpes Simplex virus VP16 transcription factor.

A) Illustration of the protein domains used in this study. ACID, activator interaction domain. TAD and AD, transcription activation domains. BD, A.thaliana Med25-ACID-binding domain. B) GST pull-down assay using purified recombinant proteins. aMed25-ACID was incubated with glutathione beads pre-bound to GST-VP16-TADs and the proteins were visualized by immunoblotting with anti-GST and anti-Med25 antibodies. Lane 1, aMed25-ACID (input); Lane 2, GST-VP16-TADn (input); Lane 3, GST-VP16-TAD (input). Lane: 4, aMed25-ACID (bound); Lane 5, aMed25-ACID + GST-VP16-TADn; Lane 6, aMed25-ACID + GST-VP16-TAD (bound). The input lanes represent 25% of the load used for each pull-down experiment. C) GST pull-down assay using recombinant proteins. GST-VP16-TADs were pre-bound to glutathione beads and incubated with hMed25-ACID (18 kDa). Proteins were separated on a 15% SDS-PAGE and stained with Coomassie blue. Lane 1, hMed25-ACID (input); Lane 2, GST-VP16-TADn (input); Lane 3, hMed25-ACID (bound); Lane 4, hMed25-ACID + GST-VP16-TADn (bound); Lane 6, GST-VP16-TAD (input); Lane 7, hMed25-ACID(bound); Lane 8, hMed25-ACID + GST-VP16-TAD (bound). D) GST pull-down assay to study competition between binding of Dreb2a_168-335 and VP16-TAD to aMed25-ACID. Dreb2a_168-335 and aMed25 were pre-incubated and then added to VP16-TADn bound to GST-beads. Lane 1, aMed25-ACID (input); Lane 2, GST-VP16-TADn (input); Lane 3, aMed25-ACID + GST-VP16-TADn (bound), Lane 4, aMed25-ACID pre-incubated with Dreb2a_168-335 + GST-VP16-TADn (bound). The input lanes represent 25% of the load used for each pull-down experiment.

Figure 2. Binding kinetics for VP16-TADn and VP16-TAD to A.thaliana and human Med25-ACIDs.

A) SPR sensogram for association and dissociation of hMed25 to GST-VP16-TADn. B) SPR sensogram for association and dissociation of aMed25 to GST-VP16-TADn. C) Binding curves plotted from the sensograms in A and B. The dissociation constants (K_D) are indicated. D) SPR sensograms for association and dissociation of hMed25 to GST-VP16-TAD. E) SPR sensograms for association and dissociation of aMed25 to GST-VP16-TAD. F) Binding curves plotted from the sensograms in D and E. The dissociation constants (K_D) are indicated. Green curves represent aMed25 and blue curves represent hMed25.

Figure 3. NMR studies of interactions between Dreb2a and hMed25-ACID or aMed25-ACID.

A) ¹H,-¹⁵N HSQC spectra of hMed25-ACID in the absence (blue) and in presence of 0.2 (cyan), 0.4 (green), 0.6 (yellow), 1 (orange) or 2 equivalents (red) of Dreb2a_168-335. Inset shows an intermediate exchange shift of a resonance corresponding to residue Q451 with a curved-like shift. B) Chemical shift changes (Δδ) obtained from NMR experiments shown in (A) plotted against the residue number of hMed25-ACID. The position of the seven β-strands and three α-helices is indicated under the residue numbers. Residues undergoing major chemical shift changes (Δδ>0.10 ppm) are labeled with the respective residue number and asterisks indicate residues which have been previously reported to be part of the interaction surface with VP16-TAD , . Dotted line corresponds to the threshold value of 0.10 (two chemical shift standard deviations). C) NMR structure of hMed25-ACID (PDB ID 2XNF) . Residues undergoing significant chemical shifts upon addition of Dreb2a_168-335 from figure (B) are enlarged and highlighted in gold and labeled with residue name and number. Resonances which are broadened beyond detection by interaction with Dreb2a_168–335 are colored in light yellow. The seven β-strands are indicated in the left view. D) Comparison of the fraction of peaks which are affected to differing degrees by interaction of Dreb2a_168–335 with Med25-ACID proteins, based on spectra presented in figure 3A and 3E. Color coding indicates peaks which are unaffected (blue), broadened beyond detection (light yellow), Δδ >0.1 (gold), Δδ <0.1 (white). E) ¹H,-¹⁵N HSQC spectra of aMed25-ACID in the absence (blue) and in presence of 0.2 (green), 0.5 (yellow), 1 (orange) or 2 equivalents (red) of Dreb2a_168–335. Inset 1 shows a cross-peak illustrating fast exchange with a curved-like shift. Inset 2 shows fast exchange shifts of two resonances (indicated by arrows). F) Similar plot as (B) but using data obtained from NMR experiments showed in (E). Δδ was plotted against the number of peaks corresponding to the number of residues of aMed25-ACID. No peak assignment is available for aMed25-ACID, therefore an identification of residues undergoing significant chemical shift changes is not possible.

Figure 4. Complex formation of Med25 and Dreb2a studied by ITC.

A) ITC profile corresponding to the binding of hMed25-ACID/Dreb2a_168–335 (blue, triangles). B) ITC profile corresponding to the binding of aMed25-ACID/Dreb2a_168–335 (green, squares). 200 µM Dreb2a_168–335 (in the syringe) was titrated into 20 µM hMed25-ACID (A) or 20 µM aMed25-ACID (B) in the reaction chamber at 25°C. The plots in the lower panels in A and B are integrated heats from raw data (upper panels) as a function of the molar ratio of Dreb2a/Med25-ACID. The binding curves were fitted using a single site binding model (Origin, MicroCal). The thermodynamic data obtained from the fitting are presented in figure (C) and Table 1. C) Histograms of the binding energetics from the two binding events in (A and B) showing the differences in enthalpic and entropic contributions from each complex formation. hMed25-ACID/Dreb2a_168–335 (blue) and aMed25-ACID/Dreb2a_168–335 (green). Change in free binding energy (ΔG), change in binding enthalpy (ΔH) and change in entropy factor (ΔTS).