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. 2009 Mar 17;48(10):2115-24.
doi: 10.1021/bi802055v.

Mapping the Interactions of the p53 Transactivation Domain With the KIX Domain of CBP

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Free PMC article

Mapping the Interactions of the p53 Transactivation Domain With the KIX Domain of CBP

Chul Won Lee et al. Biochemistry. .
Free PMC article

Abstract

Molecular interactions between the tumor suppressor p53 and the transcriptional coactivators CBP/p300 are critical for the regulation of p53 transactivation and stability. The transactivation domain (TAD) of p53 binds directly to several CBP/p300 domains (TAZ1, TAZ2, NCBD, and KIX). Here we map the interaction between the p53 TAD and the CBP KIX domain using isothermal titration calorimetry and NMR spectroscopy. KIX is a structural domain in CBP/p300 that can simultaneously bind two polypeptide ligands, such as the activation domain of MLL and the kinase-inducible activation domain (pKID) of CREB, using distinct interaction surfaces. The p53 TAD consists of two subdomains (AD1 and AD2); peptides corresponding to the isolated AD1 and AD2 subdomains interact with KIX with relatively low affinity, but a longer peptide containing both subdomains binds KIX tightly. In the context of the full-length p53 TAD, AD1 and AD2 bind synergistically to KIX. Mapping of the chemical shift perturbations onto the structure of KIX shows that isolated AD1 and AD2 peptides bind to both the MLL and pKID sites. Spin-labeling experiments show that the complex of the full-length p53 TAD with KIX is disordered, with the AD1 and AD2 subdomains each interacting with both the MLL and pKID binding surfaces. Phosphorylation of the p53 TAD at Thr18 or Ser20 increases the KIX binding affinity. The affinity is further enhanced by simultaneous phosphorylation of Thr18 and Ser20, and the specificity of the interaction is increased. The p53 TAD simultaneously occupies the two distinct sites that have been identified on the CBP KIX domain and efficiently competes for these sites with other known KIX-binding transcription factors.

Figures

Figure 1
Figure 1
Sequence of the N-terminal transactivation domain of p53. The regions that form helical structure in complexes with MDM2 (52) and replication protein A (53) are boxed. The regions corresponding to the AD1 and AD2 peptides used in this work are indicated by black bars.
Figure 2
Figure 2
1H-15N HSQC spectra of p53(13-61) and KIX. (a) 1H-15N HSQC spectra of [15N]p53(13-61) free (black) and in the presence of 2 fold excess unlabeled KIX (red). (b) 1H-15N HSQC spectra of [15N]KIX free (black) and in the presence of 2 fold excess unlabeled p53(13-61) (red). All residues are labeled on the bound (1:2 mole ratio) peaks. Cross peaks from the two tryptophan side-chains of p53 are shown in the inset.
Figure 3
Figure 3
Secondary 13Cα chemical shifts and weighted average chemical shift changes of p53(13-61). (a) 13Cα shifts calculated by subtraction of published random coil values from the experimental 13Cα chemical shifts for free p53(13-16) (black dots) and KIX bound p53(13-16) (red dots). (b) Histogram showing weighted average chemical shift changes Δδ(N,H)av(=[(ΔδHN)2+(ΔδN5)2], where ΔδHN and ΔδN correspond to the differences in amide 1H and 15N chemical shifts between the free and bound states) for p53 amide resonances caused by binding to KIX.
Figure 4
Figure 4
Histogram showing weighted average chemical shift changes for KIX amide resonances between free KIX and in the 1:1 complexes with (a) p53(13-61), (b) p53(14-28), and (c) p53(38-61).
Figure 5
Figure 5
Binding sites of p53 TAD on KIX. Weighted average chemical shift differences Δδ(N,H)av of KIX amide resonances (Fig. 4a) between free KIX and in complex with p53(13-61) are mapped onto the surface of KIX in the ternary KIX:MLL:c-myb complex (64) (PDB 2AGH), using colors to indicate changes in chemical shift greater than 2 × standard deviation from the mean (SD) (red), between 1 and 2 × SD (magenta), between mean and 1 × SD (orange), and between half of the average chemical shift difference ( Δδav/2) and the average chemical shift difference ( Δδav) (yellow). The polypeptide backbones of the bound MLL and c-Myb are shown in blue and green, respectively. The figure was prepared using MOLMOL (65).
Figure 6
Figure 6
Selected HSQC titration curves showing the average of the 15N and 1H chemical shift changes Δδ(N,H)av(=[(ΔδHN)2+(ΔδN5)2] as a function of concentration ratio for the titration of 15N labeled KIX with (a) the AD1 peptide p53(14-28), (b) the AD2 peptide p53(38-61), and (c) p53(13-61). The left three plots show the curves corresponding to the MLL binding site (residues 614, 616, 620-626, 631, 666, 669, and 670), while the right three plots show the curves corresponding to the pKID/c-Myb binding site (residues 595, 596, 600, 608, 609, 650, 652, 654, 655, 657, 661, and 663). Values of Δδ(N,H)av are plotted as symbols and the continuous lines show the curves fitted semi-globally (a, b) or globally (c) to a one-site binding model. The complete set of curves that were used to obtain the global and semi-global fits are shown in Supplementary Figure S8.
Figure 7
Figure 7
Broadening of KIX resonances by spin labeled p53 peptides. The histograms show the experimental intensity ratios (I = Ipara / Idia) for each residue with an adequately resolved cross peak in the 1H-15N HSQC spectrum of 15N KIX in complex with (a) N-terminal spin labeled p53(13-61) (black bars) and C-terminal spin labeled p53(13-61) (red bars) and (b) N-terminal spin labeled phosphorylated p53(13-57)pT18pS20 (black bars) and C-terminal spin labeled phosphorylated p53(13-57)pT18pS20 (red bars). An intensity ratio of 1 indicates no effect of the spin label on an amide proton. Residues for which quantitation was not possible due to HSQC cross peak overlap are indicated with stars (*). The location of the helices (310 helix: G1, G2 and α-helix: α1, α2 and α3) in the structure of KIX is shown schematically between the plots.
Figure 8
Figure 8
Mapping of broadening effects of spin-labeled p53(13-61) and spin-labeled phosphorylated p53(13-57)pT18pS20 on the surface of the KIX domain. The location of residues whose amides show broadening upon binding are indicated by color coding based upon the ratio of amide signal in the paramagnetic sample versus diamagnetic sample (Ipara/Idia): red = < 0.1, orange = 0.1–0.3, yellow = 0.3–0.5, blue = >0.5, and white = residues with overlapped HSQC cross peaks. The polypeptide backbones of the bound MLL and c-Myb are shown in blue and green, respectively. (a) N-terminal spin labeled p53(13-61), (b) C-terminal spin labeled p53(13-61), (c) N-terminal spin labeled p53(13-57)pT18pS20, (d) C-terminal spin labeled p53(13-57)pT18pS20. The figure was prepared using MOLMOL (65).
Figure 9
Figure 9
Alignment of the amino acid sequences of KIX binding proteins. Conserved amino acids are colored according to type: hydrophobic residues (A, V, I, L, M) are shown in shade of yellow, positively charged residues (R, K) in blue, negatively charged residues (D, E) in red.

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