S100P Interacts with p53 while Pentamidine Inhibits This Interaction

Abstract

S100P, a small calcium-binding protein, associates with the p53 protein with micromolar affinity. It has been hypothesized that the oncogenic function of S100P may involve binding-induced inactivation of p53. We used ¹H-¹⁵N HSQC experiments and molecular modeling to study the molecular interactions between S100P and p53 in the presence and absence of pentamidine. Our experimental analysis indicates that the S100P-53 complex formation is successfully disrupted by pentamidine, since S100P shares the same binding site for p53 and pentamidine. In addition, we showed that pentamidine treatment of ZR-75-1 breast cancer cells resulted in reduced proliferation and increased p53 and p21 protein levels, indicating that pentamidine is an effective antagonist that interferes with the S100P-p53 interaction, leading to re-activation of the p53-21 pathway and inhibition of cancer cell proliferation. Collectively, our findings suggest that blocking the association between S100P and p53 by pentamidine will prevent cancer progression and, therefore, provide a new avenue for cancer therapy by targeting the S100P-p53 interaction.

Keywords: 1H-5N HSQC spectrum; HADDOCK program; S100P; biomolecular docking; p53-TAD (73 amino acids); protein-protein interactions.

Figure 1

The overlaid of two HSQC spectra. The free ¹⁵N labeled p53^1-73 spectrum was showed in red. The mix of the p53^1-73 (with ¹⁵N labeled) and unlabeled S100P with 1:1 ratio was showed in blue. The cross peaks labeled with a box in this figure were the residues in p53^1-73 domain that interacted with S100P.

Figure 2

Analysis of the free S100P and S100P-p53^1-73 complex using 2D NMR at a 1:1 binding ratio. (A) The overlaid 2D [¹H-¹⁵N]-HSQC spectra highlight the spectral changes (showed with boxes) of the uniformly ¹⁵N-labeled S100P alone (shown in red) and ¹⁵N-labeled S100P upon binding to the unlabeled p53^1-73 (shown in blue). (B) These boxed cross-peak residues (Figure 2A) were colored in orange and unbound residues were colored in blue on the cartoon structure of S100P using the PyMOL program.

Figure 3

The dissociation constant (K_d) measured using the specified residues found in ¹⁵N S100P titrations with unlabeled p53^1-73, and the overall average K_d was 4.86 μM indicated with a broken line.

Figure 4

Analysis of the free S100P and S100P–pentamidine complex using 2D NMR at a 1:1 binding ratio. (A) The structural formula of pentamidine. (B) Sphere structure presentation of pentamidine. (C) The overlaid 2D [¹H-¹⁵N]-HSQC spectra highlight the spectral changes (showed in boxes) of the uniformly ¹⁵N-labeled S100P alone (shown in red) and ¹⁵N-labeled S100P upon binding to unlabeled pentamidine molecules (shown in blue). (D) These selected cross-peak residues (boxed in Figure 4C) were colored in red and unbound residues were colored in blue on the cartoon structure of S100P using the PyMOL program.

Figure 5

The dissociation constant (K_d) measured using the specified residues found in pentamidine titrations with ¹⁵N S100P HSQC, and the overall average K_d was 48.74 μM as indicated with a broken line.

Figure 6

Two complex structures S100P-p53^17-29 and S100P-pentamidine were superimposed. (A) The S100P-p53^17-29 complex is shown in blue (S100P) and purple (p53^17-29), and (B) the S100P-pentamidine complex is shown in blue (S100P) and yellow (pentamidine). (C) the overlapping structures of (A,B). (D) 90° angle rotation of (C). Figure 6D, pentamidine clearly blocked the binding between S100P (blue) and p53^17-29 (purple).

Figure 7

The effects of pentamidine on cell proliferation and p53 re-activation in ZR-75-1 breast cancer cells. ZR-75-1 cells were treated with indicated concentrations of pentamidine for 48 h. (A) Cell proliferation was determined by WST-1 assay. The bar diagram presented the relative folds of cell numbers in treated group (orange bars) comparing to that of untreated control group (blue bar). The values on the top of the bars were mean of three replicates. (B) Cell lysate was extracted from each treatment, and separated by SDS-PAGE, followed by Western blotting of p53, p21, and β-actin proteins. Beta-actin was a housekeeping protein used as the internal control of each treatment.