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Structure of the VP16 transactivator target in the Mediator - PubMed

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Structure of the VP16 transactivator target in the Mediator

Alexander G Milbradt et al. Nat Struct Mol Biol. 2011 Apr.

Abstract

The human Mediator coactivator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 subunit is a critical target of the potent herpes simplex 1 viral transcriptional activator VP16. Here we determine the solution structure of the MED25 VP16-binding domain (VBD) and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a β-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of overexpressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms.

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Figures

Figure 1
Figure 1

VP16-activated transcription. VP16, OCT-1 and HCF-1 form a VP16-induced complex. The VP16 transactivation domain (TAD) interacts with the MED25 VP16 binding domain (VBD) to recruit ARC/Mediator and activate transcription.

Figure 2
Figure 2

Solution structure of the MED25 VBD determined by NMR. (a) The 25 lowest energy structures are shown overlaid using the secondary structure elements in side-by-side stereoview. The bundles are displayed 2.4 inches apart. (b) Cartoon drawing of MED25 VBD; the seven β-strands forming the barrel and the three ⟨-helices are depicted in red and blue respectively. The long α3 helix docks on the barrel by making close contact with β5, β6 and α1. Helix α2 caps the barrel from one side. A color-coded electrostatic surface potential shows a negative patch surrounded by areas of positive potential with a hydrophobic furrow in the center (b, middle panel), highlighted by arrows.

Figure 3
Figure 3

MED25 VBD adopts a rare seven-strand β-barrel fold. Cartoon drawing of MED25 VBD accompanied by three structural homologues found using DALI: Two β-barrel-domains from the KU70-KU80 complex (PDB 1JEQ26) and the SPOC (PDB 1OW125) domain of SPEN. The three homologous structures exhibit a different topology and lack the C-terminal helix present in MED25 VBD. The three MED25 VBD homologues are shown from left to right in decreasing degree of structural homology.

Figure 4
Figure 4

Interaction of VP16 TADn with MED25 VBD. (a) Overlay of 1H-15N-HSQC spectra of VP16 TADn alone (blue) and in the presence of 1.3 equivalents of unlabeled MED25 VBD (red). While the NMR signals of MED25 VBD in the bound state (b, red signals) still show reasonable line-width and intensity, the signals of the MED25-binding site of VP16 TADn show extensive chemical exchange line broadening and attenuation, in particular for far-shifted signals (circled peaks with assignment indicated). (b) Overlay of 1H-15N-HSQC spectra of MED25 VBD alone (blue) and in the presence of 1.3 equivalents of unlabeled VP16 TADn (red). (c) Changes in the chemical shifts of the amide proton and nitrogen atoms of VP16 TADn upon binding of MED25 VBD were plotted against the residue number. Amide resonances circled in (a) are highlighted by arrows. (d) Changes in the chemical shifts of the amide proton and nitrogen atoms of MED25 VBD upon binding of VP16 TADn were plotted against the residue number. Residues wherein amide signals experienced changes of the chemical shifts greater than 0.4 ppm are highlighted by arrows. (e) A cartoon representation of MED25 VBD shows the clustering of the residues experiencing chemical shifts changes greater than 0.4 ppm (drawn with space filling spheres) upon interaction with VP16 TADn in or near the hydrophobic furrow. The changes are clustered on β3, β5, β6 and α3. (f) Residues Ile453, Leu458, ALa495, Cys497, Val510, Met512, Phe533, Ile537, and Ile541 of MED25 VBD form a central hydrophobic pocket with in the large hydrophobic furrow. Several residues in or near this pocket showed the most pronounced chemical shift changes upon binding of VP16 TADn (Fig. 4e).

Figure 5
Figure 5

Functional studies of mutant MED25 VBD. (a) VP16 full-length TAD-pull-down assays with MED25 VBD mutants. K447E, Q451E, H499E and K545E-mutated MED25 VBD exhibit weak or no binding to VP16 TAD (marked with an arrow). (b) While wild-type MED25 VBD acts in a dominant negative fashion to inhibit Gal4p-VP16 full-lenth TAD-dependent transcription, the K447E, Q451E, H499E and K545E-mutated MED25 VBD are incapable of inhibiting VP16-mediated transcription (***: p<0.01, t-test, one tailed, error bars represent s.d.). (c) The MED25 VBD Q451E mutation on β3 adjacent to the hydrophobic pocket disrupts binding of VP16 TADn to MED25 VBD. 1H-15N-HSQC spectra of free VP16 TADn (left panel), at 1:1.3 excess of wild-type MED25 VBD (middle panel) and with 1:1.5 excess of Q451E MED25 VBD (right panel) show that VP16 TADn only loosely binds, as seen by minor chemical shift changes, to mutant MED25 VBD without adopting a folded conformation. All far-shifted signals caused by the addition of wild-type MED25 VBD (middle panel, circled in red with assignment indicated) are missing when the mutant MED25 VBD is added to 15N-labeled VP16 TADn (right panel).

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