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. 2018 Feb;27(2):523-530.
doi: 10.1002/pro.3344. Epub 2017 Dec 8.

Evidence for allosteric effects on p53 oligomerization induced by phosphorylation

Petr Muller  1 , Juliana M Chan  2   3 , Oliver Simoncik  1 , Miroslav Fojta  4 , David P Lane  2 , Ted Hupp  1   5 , Borivoj Vojtesek  1
Affiliations

Evidence for allosteric effects on p53 oligomerization induced by phosphorylation

Petr Muller et al. Protein Sci. 2018 Feb.

Abstract

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)-binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the "activating" C-terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked-immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild-type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the "unfolded" conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.

Keywords: CK2; allosteric regulation; conformational change; oligomerization; p53; phosphorylation; protein conformation; protein folding.

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Figures

Figure 1
Figure 1
Samples of purified p53 protein were phosphorylated in vitro by CK2 and ATP. (A) Samples were phosphorylated in vitro, subjected to electrophoresis on a 4–16% (w/v) SDS‐polyacrylamide gel, and visualized by immuno‐blotting with DO‐1 and FP3 monoclonal antibodies at 1 μg/mL. (B) Samples were phosphorylated in vitro and titrated 10 times in a NP40 buffer for two‐site ELISA detection. Samples were captured by DO‐1 monoclonal antibody and subsequently detected with FP3‐HRP conjugated antibody. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP.
Figure 2
Figure 2
Phosphorylated p53 forms oligomers and can be detected by two‐site ELISA. (A) Schematic of two‐site ELISA detection of p53 tetramers. In the monomeric state, the N‐terminal region of p53 is captured and blocked by DO‐1. The addition of the kinase and phosphate donor pair causes p53 to form tetramers. These oligomers have exposed N‐terminal regions which can be detected by DO‐1‐HRP conjugated antibodies. (B) Samples were phosphorylated in vitro and serially diluted 10 times in NP40 buffer. Samples were captured by DO‐1 monoclonal antibody and subsequently detected with DO‐1‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP. (C) The samples were analyzed using blue native electrophoresis. The proteins transferred on membranes by western blotting were detected by DO1 antibody, phosphorylated Ser392 was detected by FP3 monoclonal antibody. Arrows labeled M, D, T indicate monomers dimers and tetramers, respectively.
Figure 3
Figure 3
Phosphorylated p53 shows increased reactivity with conformation‐specific PAb1620 monoclonal antibodies. Samples were phosphorylated in vitro and serially diluted in NP40 buffer. Samples were captured with PAb1620 monoclonal antibodies and subsequently detected with DO‐1‐HRP conjugate antibody. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP.
Figure 4
Figure 4
Detection of p53 denaturation. (A) Samples were phosphorylated in vitro and serially diluted for two‐site ELISA detection. Samples were captured with DO12 monoclonal antibody and subsequently detected with DO‐1‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against FP3‐HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP. (B) Same conditions as (A) except that capture antibody used was PAb240. (C) Phosphorylated p53 was denatured and tested for reactivity with two conformation‐specific monoclonal antibodies, DO12 and PAb240. Samples were phosphorylated in vitro and serially diluted 10 times for two‐site ELISA detection. One sample was denatured by 15 mM of SDS for 30 min at 65°C. Samples were captured with DO12 or PAb240 monoclonal antibodies and subsequently detected with DO‐1‐HRP conjugate. In the bar chart, HRP activity (absorbance at 450 nm) is represented as a percentage value on the y‐axis. The absorbance values of the p53 + SDS samples are taken as 100% denatured p53 protein for reference.
Figure 5
Figure 5
Different CK2 concentrations affect p53 phosphorylation status. (A) Samples were phosphorylated in vitro, subjected to electrophoresis on a 4–16% (w/v) SDS‐polyacrylamide gel and visualized by immunoblotting with DO‐1 and FP3 monoclonal antibodies at 1 μg/mL. The graph shows the relative density of bands detected by phospho‐specific antibody FP3. (B) Samples were phosphorylated in vitro and serially diluted for two‐site ELISA detection. Samples were captured with DO‐1 monoclonal antibody and subsequently detected with FP3‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + ATP/CK2 500 U; (▲) p53 + ATP/CK2 50 U; (×) p53 + ATP/CK2 5 U.
Figure 6
Figure 6
CK2 concentration affects p53 oligomerization. CK2 forms complexes with phosphorylated p53. (A) Samples were phosphorylated in vitro and serially diluted for two‐site ELISA detection. Samples were captured with DO‐1 monoclonal antibody and subsequently detected with DO‐1‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (B) Samples were prepared identically and were detected by anti‐CK2 β rabbit monoclonal antibody and secondary swine‐anti‐rabbit polyclonal antibodies conjugated to HRP.
Figure 7
Figure 7
CK2 binding to p53 blocks the Bp53‐10 epitope within the C‐terminal region of p53. p53 was incubated with either 500 units (A) or 5 units (B) of CK2 and ATP. Samples were phosphorylated in vitro and serially diluted ten times in NP40 buffer. Samples were captured with DO‐1 monoclonal antibody and subsequently detected with Bp53‐10‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as a percentage of the maximal value obtained.
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