Ultraslow oligomerization equilibria of p53 and its implications

Abstract

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 wildtype (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°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, wt2mut2, and mut4, 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.DNA, wt2mut2.DNA, and mut 4.DNA, 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.mass spectrometry ͉ protein-protein interactions ͉ slow association ͉ tetramerization domain T he tumor suppressor p53 is a transcription factor that is inactivated by mutation in some 50% of human cancers (1, 2). p53 consists of an unstructured N-terminal domain (residues 1-94) which points away from the rest of the protein (3), a folded core domain (residues 94-292), a linker to the tetramerization domain (residues 325-355) and an unstructured C-terminal domain . Nearly all of the mutations reside in its DNA-binding domain. Mutations can be either 'contact,' which remove residues that interact with DNA and lower affinity, or 'structural,' which destabilize the protein, sometimes with concomitant conformational changes (4). The transcriptionally active form of p53 is a homo-tetramer, linked via its tetramerization domains. Since most cancer mutations are located outside the tetramerization domain, in the core domain, the mutant (mut) proteins retain wild-type (wt) ability to form tetramers. Consequently the formation of hetero-tetramer complexes between wt and mut p53 is observed both in vivo and in vitro (5). It is thought that formation of hybrids between the wt and the mut protein is the cause of the trans-dominant effect of the mut over the wt in heterozygous cells (dominant-negative effect), either following somatic mutation or from birth as in the case of Li-Fraumeni syndrome (5, 6). In such events, the chances of initiating cancer are increased (7), and the p53 het...

A single-molecule characterization of p53 search on DNA

“…This estimate is consistent with about an hour for initial expression of downstream genes (25). Moreover this reasoning suggests that the latent p53 with a lifetime of about 20 min and its slow oligomerization kinetics (26,27) is unlikely to yield significant occupancy in tetrameric form at hundreds of target promoters. The search, however, is fast enough to allow a long-lived activated form (lifetime of ∼200 min) (28,29) to bind most of target promoters.…”

“…The single-molecule experiments were done within less than 1 h from the time of dilution. Due to the slow kinetics of the tetramer-dimer transition (26), all constructs are assumed to be in the tetrameric form during the single-molecule experiment.…”

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

“…The dynamics of the L7/L12 proteins were investigated using an MS strategy that has proven successful to elucidate the subunit exchange dynamics of other protein complexes (30)(31)(32)(33). Specifically, ribosomes are uniformly labeled with stable isotopes ( 13 C and 15 N) to provide sufficient mass differences to resolve heterocomplexes formed with wild-type ribosomes.…”

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

“…Additionally, the rapid analysis possible with ESI-MS makes it convenient for following subunit exchange in real time [38]. Despite these benefits, only a small number of studies applying ESI-MS to multimeric protein subunit exchange have been reported [39][40][41][42][43][44][45][46][47][48].…”