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. 2012;7(12):e52906.
doi: 10.1371/journal.pone.0052906. Epub 2012 Dec 31.

Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex

Ojore Oka  1 , Lorna C Waters, Sarah L Strong, Nuvjeevan S Dosanjh, Vaclav Veverka, Frederick W Muskett, Philip S Renshaw, Karl-Heinz Klempnauer, Mark D Carr
Affiliations

Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex

Ojore Oka et al. PLoS One. 2012.

Abstract

The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (K(d) ~1.0-10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (~1200 Å(2)). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.

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Conflict of interest statement

Competing Interests: Limited support for the work reported in the paper was provided by UCB Celltech Ltd through support for the Research Fellow position held by Vaclav Veverka. However, this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Schematic representations of the organisation of the functional regions and domains of human B-Myb and p300.
Panel A shows the positions of functional domains in the transcriptional coactivator p300, as well as a partial list of proteins that bind to the CH3/E1A-binding region. Panel B illustrates the tripartite functional organisation of the B-Myb protein, which contains an N-terminal DNA binding region (DBD) formed by three highly homologous domains (R1, R2 and R3), a central transactivation domain (TAD), and towards the C-terminus a highly conserved region (CR) and negative regulatory domain (NRD).
Figure 2
Figure 2. Far UV circular dichroism analysis of the B-Myb TAD and p300 TAZ2 domain.
Panels A illustrates a typical far UV circular dichroism (CD) spectra obtained for the B-Myb TAD. Panel B shows representative intrinsic tryptophan fluorescence emission spectra obtained for the B-Myb TAD in the absence (i) and presence (ii) of an approximately three-fold molar excess of p300 TAZ2. In panel C far UV CD spectra of TAZ2 are shown in the absence (i) and presence (ii) of a molar excess of EDTA over Zn2+ ions.
Figure 3
Figure 3. Comparison of NMR assignments and secondary structures for the TAZ2 domains of CBP and p300.
Panel A summarises the combined differences in backbone amide (15N and 1H), CO and Cα chemical shifts for equivalent residues in the TAZ2 domains of CBP and p300. To compensate for the increased chemical shift range of 15N and 13C compared to 1H, the combined change was calculated as (Δ1HN+(Δ15N × 0.2)+(Δ13Cα × 0.1)+(Δ13CO × 0.35))/4. In a very few cases where some of the chemical shifts were not available, the sum of the chemical shift changes was divided by the number of available shift differences. Panel B shows an alignment of the very closely related TAZ2 sequences from CBP and p300. Conservative substitutions are highlighted in an open box and non-conservative highlighted in grey. The black bars shown indicate the positions of the helices in CBP TAZ2 , whilst the white bars represent the positions of the helices in p300 TAZ2, which were identified by analysis of the backbone resonance assignments using the chemical shift index method .
Figure 4
Figure 4. Binding of the B-Myb TAD to the TAZ2 domain of p300.
The SDS-PAGE gel shown in panel A illustrates the analysis of a typical pull down experiment using immobilised GST-B-Myb TAD as bait and a slight excess of p300 TAZ2 as the potential interaction partner. p300 TAZ2 was loaded onto a glutathione agarose that had been preloaded with a 0.5 ml sample of GST-B-Myb TAD (32.5 µM), the column was washed with 8 column volumes of binding buffer prior to elution. Lane M contains molecular weight markers, lane 1 contains the GST-B-Myb load, lane 2 the p300 TAZ2 load, lanes 3–8 are consecutive washes, and lanes 9 to 11 are consecutive elution fractions. The SDS-PAGE gel shown in panel B shows the results of a control pull-down assay in which p300 TAZ2 was loaded onto the column in the presence of GST alone. The samples loaded on the gel are identical to those described in panel A, except lane 1 contains the GST load.
Figure 5
Figure 5. Identification of the B-Myb TAD binding site on p300 TAZ2.
Panel A shows an overlay of two 15N/1H HSQC spectra of 15N labeled p300 TAZ2 (100 µM) acquired in the absence (red) or presence of equimolar unlabelled B-Myb TAD (black). The arrows highlight a number of TAZ2 signals which show significant shifts on interaction with the B-Myb TAD. Panel B contains a histogram summarizing the minimal chemical shift changes observed for backbone amide signals of p300 TAZ2 on binding to B-Myb TAD, with gaps corresponding to proline residues (1727, 1756, 1780, 1802 and 1804) or non-observable backbone amides. The combined amide proton and nitrogen chemical shift difference (Δδ) was defined according to the calculation formula image where αN is a scaling factor of 0.2 required to account for differences in the range of amide proton and nitrogen chemical shifts. The reported positions of the helices in CBP TAZ2 (blue bars, [30]), together with those determined for p300 TAZ2 (yellow bars), are shown above the histogram. Panel C shows a ribbon representation of the backbone structure of the TAZ2 domain of CBP and panel D a contact surface view in the same orientation. In panel E the surface view of CBP TAZ2 has been rotated by 180° about the y axis. The contact surfaces have been coloured according to the magnitude of the minimal shifts induced in backbone amide resonances of equivalent residues in p300 TAZ2 by binding of the B-Myb TAD. Residues that showed a minimal shift change of less than 0.075 ppm are shown in white, over 0.15 ppm in red, and between 0.075 and 0.15 ppm are coloured according to the level of the shift on a linear gradient between white and red. No chemical shift perturbation data could be obtained for the residues shown in yellow.
Figure 6
Figure 6. Potential amphipathic helices in the B-Myb TAD.
Panels A and B show helical wheel representations of the regions of the B-Myb TAD predicted to form amphipathic helices, charged residues are underlined and polar residues shown in italics. The positions of the helical regions were predicted using the programme PSIPRED , .
Figure 7
Figure 7. Comparison of the B-Myb, STAT1, E1A and p53 transactivation domain binding sites on p300/CBP TAZ2.
Panel A shows a contact surface view of CBP TAZ2 (top) with the location of the B-Myb TAD binding site on p300 TAZ2 highlighted as described in figure 5. For comparison, the structures of STAT1 TAD-CBP TAZ2 (row 2; PDB code 2KA6), E1A CR1-CBP TAZ2 (row 3; PDB code 2KJE) and p53 TAD1-p300 TAZ2 (row 4 PDB code 2K8F) are shown in the same orientation , , , with the TAZ2 domain shown as a contact surface and the STAT1 TAD, E1A CR1 and p53 TAD1 as a ribbon representation of their backbone conformation. Only the well defined residues of STAT1 (721–750), E1A (53–83) and p53 (9–31) that contact TAZ2 are shown in the figure. The views in panels B and C are rotated about the y axis by 90° and −90° compared to panel A. Panel D shows the structure of STAT1 TAD-CBP TAZ2, in the same orientation shown in panel A, with the TAZ2 domain shown as a contact surface and STAT1 TAD as a ribbon representation of the domain. TAZ2 residues are coloured on the basis of residue type, with basic amino acids in blue (Arg, Lys and His), acidic in red (Asp and Glu), polar in orange (Ser, Thr, Asn and Gln), cysteine in green and hydrophobic in white (Trp, Phe, Tyr, Ala, Val, Ile, Leu, Met, Pro and Gly).
Figure 8
Figure 8. Multiple sequence alignment of the highly homologous TAZ2 domains of p300 and CBP.
The multiple sequence alignment of the TAZ2 domain of human, mouse, western clawed frog (Xenopus tropicalis), stickleback and chicken p300, and drosophila and pond snail CBP, illustrates the high degree of sequence homology between the TAZ2 domains of a diverse range of species. Residues are coloured according to the residue type, with small and hydrophobic residues in red (AVFPMILW), acidic residues in blue (DE), basic residues in magenta (RK) and residues containing a hydroxyl, sulfhydryl or sidechain amide group in green (STYHCNQ). Glycine was also coloured in green. Consensus symbols are shown below the sequence. Residues marked with an ‘*’ were fully conserved between sequences. The symbol ‘:’ indicates conservation between groups with strongly similar properties and ‘.’ indicates conservation between groups of weakly similar properties. TAZ2 residues that were significantly shifted upon binding to B-Myb are indicated by triangles shown below the consensus. The positions of the helices in p300 TAZ2, which were identified by analysis of the backbone resonance assignments using the chemical shift index method are indicated above the sequence. The alignment was prepared using ClustalW.
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