Figure 1.
The C-terminal domain contains the folded tetramerization domain (red) flanked by two unfolded domains (cyan). TAD – transactivation domain; CTD–C-terminal domain.
Figure 2.
Screening of the peptide array for binding to p53CTD and the constructs p53Tet and p53NRD.
Peptides that interacted with p53NRD (p53 361–393) are marked in green. The peptide WS100B(81–92) that interacted with p53Tet is marked in yellow. The peptide PKCα(281–295) that bound more tightly to full-length p53CTD than to p53NRD is marked in red.
Table 1.
Binding peptides identified in the peptide array screening.
Figure 3.
Binding of peptides from the array to p53CTD.
p53CTD was titrated into a solution of fluorescein-labeled peptide and the changes in anisotropy were measured. All data were fit to a 1∶1 binding model, indicated by solid lines.
Table 2.
Binding affinities of peptides from the array to p53CTD and their effect in AUCA.
Figure 4.
Ionic strength-dependent binding of the peptides to p53CTD.
ln(Kd) is plotted vs. ln(NaCl activity) for the tightest binding peptides. The data were fit to a linear model.
Table 3.
Number of dissociated ion pairs upon peptide binding to p53CTD.
Figure 5.
Changes in chemical shifts of 15N-labeled p53CTD upon incubation with various peptides.
15N–1H HSQC NMR spectra of the protein were measured with and without peptide, and the changes in chemical shifts were calculated as (δΔ1H2+(δΔ15N/5)2)0.5. The left panel shows the chemical shift deviations for backbone amide nitrogen atoms, and the right panel shows the chemical shift deviations for side chain nitrogen atoms. Numbers on the x-axis are arbitrary serial numbers for peaks and are unrelated to residue sequence.
Figure 6.
Thermal denaturation curves of p53CTD L344A in the presence of peptides from the array.
20 µM protein was heated from 25°C to 65°C with or without 100 µM peptide. The data were fit to a sigmoidal curve describing a transition between two states. Raw data for two repeats (squares and hollow circles) are shown in the background. The dashed lines represent the averaged melting temperatures for both repeats.
Table 4.
Melting temperatures of p53CTD L344A in the presence of peptides from the array.
Figure 7.
Sedimentation profiles of FlAsH-labeled p53 (25–50 nM) measured in the presence of the peptides.
A) PKCα(271–285) had no significant effect on the profile. B) Peptides that shifted the tetramer peak. C) Concentration dependent behavior of the peptides. Sedimentation profiles were measured with different concentrations of WS100B(61–75). Similar behavior was observed for the other peptides in B (data not shown). D) Behavior of Cul7(376–390). This peptide binds p53 only weakly, and causes a shift of the tetramer peak at very high peptide concentrations. Similar behavior was observed for PARC(386–400).
Figure 8.
Binding sites for p53CTD on two PKCα domains.
A) The C2 domain of PKCα (pdb 1dsy). Residues 271–292 are colored in red. B) The catalytic domain of PKCα (pdb 3iw4). Residues 641–655 are colored in red.
Figure 9.
NMR structure (pdb 2jng) of the Cul7 CPH domain, residues 360–440.
The binding site for p53CTD is color marked as indicated in the figure. The two views are rotated∼180° with respect to each other. A) Direct view of the LDDYEE motif; B) Direct view of the DEGEF motif.