Ultraslow oligomerization equilibria of p53 and its implications.
The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wild-type (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t(1/2) = 40 min) at 37 degrees C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt(4), wt(2)mut(2), and mut(4), in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt(4)xDNA, wt(2)mut(2)xDNA, and mut(4)xDNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.
Natan
Eviatar
E
Medical Research Council Centre for Protein Engineering, Cambridge CB2 0QH, United Kingdom.
Hirschberg
Daniel
D
Morgner
Nina
N
Robinson
Carol V
CV
Fersht
Alan R
AR
D019943:Amino Acid Substitution; D001667:Binding, Competitive; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050505:Mutant Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D004247:DNA