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The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.

Bom AP, Freitas MS, Moreira FS, Ferraz D, Sanches D, Gomes AM, Valente AP, Cordeiro Y, Silva JL - J. Biol. Chem. (2009)

Bottom Line: This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH.Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0).The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.

ABSTRACT
p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.

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Overlay of wt p53 1H-15N HSQC spectra at different pH values. A, blue and red spectra show wt p53C at pH 7.2 and 5.0, respectively. Compared with pH 7.2, pH 5.0 induced a reduction in the resonance signals and a decrease in the peak dispersions. B, representation of wt p53 core domain. 1H-15N HSQC correlation spectrum of p53C at pH 5.0 was compared with the p53C spectrum at pH 7.2. Residues that presented changes in amide chemical shifts at pH 5.0 are colored in violet.
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Figure 5: Overlay of wt p53 1H-15N HSQC spectra at different pH values. A, blue and red spectra show wt p53C at pH 7.2 and 5.0, respectively. Compared with pH 7.2, pH 5.0 induced a reduction in the resonance signals and a decrease in the peak dispersions. B, representation of wt p53 core domain. 1H-15N HSQC correlation spectrum of p53C at pH 5.0 was compared with the p53C spectrum at pH 7.2. Residues that presented changes in amide chemical shifts at pH 5.0 are colored in violet.

Mentions: The lack of fixed tertiary interactions in many globular proteins results in a fluctuating ensemble of structures (50). Although these molecules are heterogeneous, valuable structural information can be acquired through nuclear magnetic resonance (NMR) measurements. In general, folded proteins have good resonance signal dispersions and sharp peaks. MG states lose resonance signals and have much less signal dispersion. MG conformational heterogeneity is a result of residual secondary and side chain interactions; therefore, cross-peaks in the 15N-1H HSQC spectrum tend to be broad or disappear completely (51). To further characterize the population of p53C molten globule structures at acidic pH, we carried out 15N-edited NMR experiments of wt and R248Q p53C at pH 7.2 and 5.0 (Fig. 5). In the native state (blue), a broad distribution of resonance signals was observed, as previously described (52). At pH 5.0, there was a reduction in peak dispersion (Fig. 5, red plot). The wt p53C HSQC dispersion at pH 7.2 was observed from 10.142 to 6.075 ppm (1H dimension), compared with 8.791 to 6.782 ppm (1H dimension) at pH 5.0. We suggest that a broad set of peaks experiences different perturbations at pH 7.2 and represents the observed differences between pH 7.2 and 5.0, based on the backbone structure of wt p53C. Residues that have 1H and 15N chemical shifts at pH 5.0 are shown in violet (Fig. 5B). Curiously, the conformational changes are spread throughout the whole p53C structure (Fig. 5). In contrast, several residues of the MG state do not change significantly compared with the native state. The R248Q mutant, classified both as a contact and structural mutant (40), presented HSQC dispersion at pH 7.2 from 10.1360 to 6.6343 ppm and at pH 5.0 from 8.793 to 6.763 ppm (supplemental Fig. S2). These results were similar to those of the wild-type protein. Based on these data, we suggest that the MG states of both proteins (wt and R248Q p53C) are very similar. Subsequently, the samples were applied to a gel filtration column to analyze whether there were conformational transitions. Our data show that wt p53C at pH 7.2 has the same elution profile before and after HSQC measurement (data not shown). After the NMR experiment at pH 5.0, about 10–15% of the protein was in an oligomeric form. This result indicates that wt p53C can adopt different oligomeric states after longer incubation times at pH 5.0, probably because the MG is a flexible state, and some of the intramonomeric interactions are replaced by intermolecular interactions.


The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.

Bom AP, Freitas MS, Moreira FS, Ferraz D, Sanches D, Gomes AM, Valente AP, Cordeiro Y, Silva JL - J. Biol. Chem. (2009)

Overlay of wt p53 1H-15N HSQC spectra at different pH values. A, blue and red spectra show wt p53C at pH 7.2 and 5.0, respectively. Compared with pH 7.2, pH 5.0 induced a reduction in the resonance signals and a decrease in the peak dispersions. B, representation of wt p53 core domain. 1H-15N HSQC correlation spectrum of p53C at pH 5.0 was compared with the p53C spectrum at pH 7.2. Residues that presented changes in amide chemical shifts at pH 5.0 are colored in violet.
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Related In: Results  -  Collection

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Figure 5: Overlay of wt p53 1H-15N HSQC spectra at different pH values. A, blue and red spectra show wt p53C at pH 7.2 and 5.0, respectively. Compared with pH 7.2, pH 5.0 induced a reduction in the resonance signals and a decrease in the peak dispersions. B, representation of wt p53 core domain. 1H-15N HSQC correlation spectrum of p53C at pH 5.0 was compared with the p53C spectrum at pH 7.2. Residues that presented changes in amide chemical shifts at pH 5.0 are colored in violet.
Mentions: The lack of fixed tertiary interactions in many globular proteins results in a fluctuating ensemble of structures (50). Although these molecules are heterogeneous, valuable structural information can be acquired through nuclear magnetic resonance (NMR) measurements. In general, folded proteins have good resonance signal dispersions and sharp peaks. MG states lose resonance signals and have much less signal dispersion. MG conformational heterogeneity is a result of residual secondary and side chain interactions; therefore, cross-peaks in the 15N-1H HSQC spectrum tend to be broad or disappear completely (51). To further characterize the population of p53C molten globule structures at acidic pH, we carried out 15N-edited NMR experiments of wt and R248Q p53C at pH 7.2 and 5.0 (Fig. 5). In the native state (blue), a broad distribution of resonance signals was observed, as previously described (52). At pH 5.0, there was a reduction in peak dispersion (Fig. 5, red plot). The wt p53C HSQC dispersion at pH 7.2 was observed from 10.142 to 6.075 ppm (1H dimension), compared with 8.791 to 6.782 ppm (1H dimension) at pH 5.0. We suggest that a broad set of peaks experiences different perturbations at pH 7.2 and represents the observed differences between pH 7.2 and 5.0, based on the backbone structure of wt p53C. Residues that have 1H and 15N chemical shifts at pH 5.0 are shown in violet (Fig. 5B). Curiously, the conformational changes are spread throughout the whole p53C structure (Fig. 5). In contrast, several residues of the MG state do not change significantly compared with the native state. The R248Q mutant, classified both as a contact and structural mutant (40), presented HSQC dispersion at pH 7.2 from 10.1360 to 6.6343 ppm and at pH 5.0 from 8.793 to 6.763 ppm (supplemental Fig. S2). These results were similar to those of the wild-type protein. Based on these data, we suggest that the MG states of both proteins (wt and R248Q p53C) are very similar. Subsequently, the samples were applied to a gel filtration column to analyze whether there were conformational transitions. Our data show that wt p53C at pH 7.2 has the same elution profile before and after HSQC measurement (data not shown). After the NMR experiment at pH 5.0, about 10–15% of the protein was in an oligomeric form. This result indicates that wt p53C can adopt different oligomeric states after longer incubation times at pH 5.0, probably because the MG is a flexible state, and some of the intramonomeric interactions are replaced by intermolecular interactions.

Bottom Line: This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH.Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0).The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.

ABSTRACT
p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.

Show MeSH
Related in: MedlinePlus