The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.
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.
Bom
Ana Paula D Ano
AP
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.
Freitas
Monica S
MS
Moreira
Flavia S
FS
Ferraz
Danielly
D
Sanches
Daniel
D
Gomes
Andre M O
AM
Valente
Ana Paula
AP
Cordeiro
Yraima
Y
Silva
Jerson L
JL
D000143:Acids / Q000737:chemistry*; D001943:Breast Neoplasms / Q000737:chemistry*; D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics
D000143:Acids; D012997:Solvents; D016159:Tumor Suppressor Protein p53; D014364:Tryptophan