Interaction of the Transactivation Domain of B-Myb with the TAZ2 Domain of the Coactivator p300: Molecular Features and Properties of the Complex

<div><p>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 Ų). 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.</p> </div

Comparison of the B-Myb, STAT1, E1A and p53 transactivation domain binding sites on p300/CBP TAZ2.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone-0052906-g005" target="_blank">figure 5</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-Wojciak1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-Ferreon1" target="_blank">[61]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-Feng1" target="_blank">[64]</a>, 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).</p

Identification of the B-Myb TAD binding site on p300 TAZ2.

<p>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 where α_N 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, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-DeGuzman1" target="_blank">[30]</a>), 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-DeGuzman1" target="_blank">[30]</a> 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.</p

Potential amphipathic helices in the B-Myb TAD.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-Bryson1" target="_blank">[71]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-Jones1" target="_blank">[72]</a>.</p

Far UV circular dichroism analysis of the B-Myb TAD and p300 TAZ2 domain.

<p>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.</p

Schematic representations of the organisation of the functional regions and domains of human B-Myb and p300.

<p>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).</p

Comparison of NMR assignments and secondary structures for the TAZ2 domains of CBP and p300.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-DeGuzman1" target="_blank">[30]</a>, 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052906#pone.0052906-Wishart1" target="_blank">[45]</a>.</p

Multiple sequence alignment of the highly homologous TAZ2 domains of p300 and CBP.

<p>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.</p

CSPs cluster at the potential FcRn_ECD diprotomer interface.

<p><b>(A)</b> CSPs in surface representation of the FcRn_ECD diprotomer crystal structure in complex with UCB-FcRn-303 (red), with the same color-coding as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006192#pbio.2006192.g005" target="_blank">Fig 5</a>. <b>(B)</b> For orientation, the FcRn_ECD crystal structure is shown in cartoon representation with β2m in green and dark grey and the α-chain molecules in blue and light grey. <b>(C)</b> The IgG and HSA interaction sites are depicted in purple and orange, respectively. The highlighted residues are discussed in the text. CSP, chemical-shift perturbation; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor; HSA, Human Serum Albumin; IgG, Immunoglobulin G.</p

Crystal structure of the compound UCB-FcRn-303 (R enantiomer) bound to FcRn_ECD.

<p><b>(A)</b> The protein crystallized as a dimer composed of two β2m (dark grey and green) and two α-chain (light grey and blue) molecules. <b>(B)</b> At the interface of β2m and the α-chain, UCB-FcRn-303 (grey) occupies a binding pocket with Glycine, Cysteine, hydrophobic (Leucine), charged (Histidine, Aspartate), and polar uncharged (Serine, Glutamine) residues. β2m, β2-microglobulin; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor.</p