Nt broadening of selected p300 TAZ2 resonances on complex formation implies

Nt broadening of selected p300 TAZ2 resonances on complex formation implies intermediate exchange on the NMR time scale between the free and bound species, which is consistent with a Kd in the low micromolar range (1?0 mM). Similar relatively low affinity interactions have recently been reported for a number of protein Title Loaded From File complexes involved in transcriptional and translational regulation [65], [69], [70], which is consistent with the requirement to form transient or dynamic complexes for effective regulation of these processes. The TAZ2 signals most perturbed by B-Myb TAD binding correspond to residues Arg1731, Leu1742, Arg1763, Thr1768, Lys1769, Gly1770, Lys1774, Thr1775, Gln1784, Ile1786, Ala1787, Cys1789, Cys1790, Tyr1791, His1792, Lys1794 and Cys1796 (figure 5B). The locations of the perturbed residues were mapped onto the surface of both the isolated CBP TAZ2 domain ([30]: PDB code 1F81: figure 5 panels D E) and the isolated p300 TAZ2 domain ([67]: PDB code 3IO2: figure S2). The majority of the shifted residues are located at the C- terminus of a2, in a29, and on the exposed face of a3. These residues form a ?large patch on the surface of TAZ2 (,1200 A2, figure 5), which is consistent with forming a contiguous binding surface for the BMyb TAD, rather than reflecting a conformational change induced by B-Myb TAD binding. Interestingly, a number ofFigure 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, Title Loaded From File 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. doi:10.1371/journal.pone.0052906.gFeatures of the B-Myb TAD-p300 TAZ2 Complexresidues for which we were unable to obtain chemical shift mapping data 1527786 (Pro1780 and Cys1801-Ile1809), including several that appear to be in conformational exchange in the isolated p300 TAZ2 domain, are located adjacent to this patch and it seems likely that some or all of these will form part of the B-Myb-binding surface. The transactivation domain (TAD) of the transcription factor STAT1 (Signal transducer and activator of transcription-1, residues 710?50) has been shown to interact with essentially the same surface of CBP TAZ2 as reported here for B-Myb TAD (figure 7) [56]. Interestingly, the core of the overlapping B-Myb TAD and STAT1 TAD binding surface on TAZ2 is absolutely conserved over a diverse range of species as is clearly evident.Nt broadening of selected p300 TAZ2 resonances on complex formation implies intermediate exchange on the NMR time scale between the free and bound species, which is consistent with a Kd in the low micromolar range (1?0 mM). Similar relatively low affinity interactions have recently been reported for a number of protein complexes involved in transcriptional and translational regulation [65], [69], [70], which is consistent with the requirement to form transient or dynamic complexes for effective regulation of these processes. The TAZ2 signals most perturbed by B-Myb TAD binding correspond to residues Arg1731, Leu1742, Arg1763, Thr1768, Lys1769, Gly1770, Lys1774, Thr1775, Gln1784, Ile1786, Ala1787, Cys1789, Cys1790, Tyr1791, His1792, Lys1794 and Cys1796 (figure 5B). The locations of the perturbed residues were mapped onto the surface of both the isolated CBP TAZ2 domain ([30]: PDB code 1F81: figure 5 panels D E) and the isolated p300 TAZ2 domain ([67]: PDB code 3IO2: figure S2). The majority of the shifted residues are located at the C- terminus of a2, in a29, and on the exposed face of a3. These residues form a ?large patch on the surface of TAZ2 (,1200 A2, figure 5), which is consistent with forming a contiguous binding surface for the BMyb TAD, rather than reflecting a conformational change induced by B-Myb TAD binding. Interestingly, a number ofFigure 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. doi:10.1371/journal.pone.0052906.gFeatures of the B-Myb TAD-p300 TAZ2 Complexresidues for which we were unable to obtain chemical shift mapping data 1527786 (Pro1780 and Cys1801-Ile1809), including several that appear to be in conformational exchange in the isolated p300 TAZ2 domain, are located adjacent to this patch and it seems likely that some or all of these will form part of the B-Myb-binding surface. The transactivation domain (TAD) of the transcription factor STAT1 (Signal transducer and activator of transcription-1, residues 710?50) has been shown to interact with essentially the same surface of CBP TAZ2 as reported here for B-Myb TAD (figure 7) [56]. Interestingly, the core of the overlapping B-Myb TAD and STAT1 TAD binding surface on TAZ2 is absolutely conserved over a diverse range of species as is clearly evident.