Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

The tumor suppressor p53 is a homotetramer of 4 × 393 residues. Its core domain and tetramerization domain are linked and flanked by intrinsically disordered sequences, which hinder its full structural characterization. There is an outstanding problem of the state of the tetramerization domain. Structural studies on the isolated tetramerization domain show it is in a folded tetrameric conformation, but there are conflicting models from electron microscopy of the full-length protein, one of which proposes that the domain is not tetramerically folded and the tetrameric protein is stabilized by interactions between the N and C termini. Here, we present methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) investigations on the full-length and separate domains of the protein with its methionine residues enriched with 13 C to probe its quaternary structure. We obtained high-quality spectra of both the full-length tetrameric p53 and its DNA complex, observing the environment at 11 specific methyl sites. The tetramerization domain was as tetramerically folded in the full-length constructs as in the isolated domain. The N and C termini were intrinsically disordered in both the full-length protein and its complex with a 20-residue specific DNA sequence. Additionally, we detected in the interface of the core (DNA-binding) and N-terminal parts of the protein a slow conformational exchange process that was modulated by specific recognition of DNA, indicating allosteric processes.transcription factor | intrinsically disordered protein I ntrinsically disordered domains are crucial, functional components of many proteins, especially those involved in cell signaling and regulation of the cell cycle (1, 2), p53 being an archetypical example of such a protein. p53 is a homotetramer of 4 × 393 residues ( Fig. S1): Residues 1-93 form the intrinsically disordered N-terminal domain (TAD), with a proline-rich region (PRR) 61-93; 94-292 form the folded core domain (p53C); 293-324 form a disordered linker; 325-353 associate to form the folded tetramerization domain (TET); and 354-393 are intrinsically disordered (CT). The large size of this protein and presence of disordered regions have so far prevented the full-length p53 from being crystallized or its high-resolution structure solved by NMR. The structures of isolated p53 domains have been extensively studied by high-resolution methods (for an overview see ref. 3; Fig. S1); however, their arrangement in the full-length protein has been a subject of controversy, and alternative models of quaternary structural arrangement have been postulated. One model of the human protein obtained from electron microscopy and small-angle X-ray scattering (SAXS) is consistent with structural studies on the isolated TET domain whereby the full-length tetramer and truncated constructs associate via the TET domain (4-6). But in a model of the murine protein, it is proposed that oligomerization occurs via contacts between N and C termini of the protein, as well as the core domains, and th...

Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

Bista

Freund

Fersht

2012

“…Tabulated values of K d are concentrations of p53 in terms of monomer required for 50% binding of labeled DNA. The apparent cooperativity, h, of binding results from the association of p53 dimers into tetramers with a K d of 20 nM (45,46). Thus, the predominant form of p53 during the measurement of weak binders (that is around the concentration for 50% binding) is the tetramer, and the binding is not cooperative (20).…”

Section: Resultsmentioning

confidence: 99%

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Arbely

Natan

Brandt

et al. 2011

Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNAðp53DBDÞ 4 or DNA ðAcK120DBDÞ 4 , indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

“…In in vitro studies, p53 first assembles into homo-dimers with a K d of ∼1 nM (8), and these dimers then come together in tetramers with a K d of ∼100 nM-1 μM (8-11). The K d of tetramerization in vitro can be lowered by specific posttranslational modifications (10)(11)(12).…”

mentioning

confidence: 99%

“…FCS is widely used in vitro to measure protein homo-oligomerization, including p53 tetramerization (4,8), but has only rarely been used in living cells for this purpose (15). FCS provides direct measurements of the intensity and brightness of fluorescent molecules (16); the intensity reports the numbers of fluorescent molecules in the volume and therefore provides a measure of total protein concentration.…”

mentioning

confidence: 99%

Activation and control of p53 tetramerization in individual living cells

Gaglia

Guan

Shah

et al. 2013

107

105

Homo-oligomerization is found in many biological systems and has been extensively studied in vitro. However, our ability to quantify and understand oligomerization processes in cells is still limited. We used fluorescence correlation spectroscopy and mathematical modeling to measure the dynamics of the tetramers formed by the tumor suppressor protein p53 in single living cells. Previous in vitro studies suggested that in basal conditions all p53 molecules are bound in dimers. We found that in resting cells p53 is present in a mix of oligomeric states with a large cell-to-cell variation. After DNA damage, p53 molecules in all cells rapidly assemble into tetramers before p53 protein levels increase. We developed a model to understand the connection between p53 accumulation and tetramerization. We found that the rapid increase in p53 tetramers requires a combination of active tetramerization and protein stabilization, however tetramerization alone is sufficient to activate p53 transcriptional targets. This suggests triggering tetramerization as a mechanism for activating the p53 pathway in cancer cells. Many other transcription factors homo-oligomerize, and our approach provides a unique way for probing the dynamics and functional consequences of oligomerization.H omo-oligomerization, the formation of a protein complex out of identical components, is extremely common in nature; in Escherichia coli it is estimated that 35% of proteins form homo-oligomers (1), with an average of four subunits per complex. In yeast and human cells many transcription factors undergo homo-oligomerization, which has been shown to be crucial for their function (2). The molecular dynamics of oligomerization have been studied for some proteins in vitro, but no study has quantified a discrete number of oligomers in a dynamic oligomerization process in live single cells. Here we focus on the homo-tetramers formed by the tumor suppressor p53 and quantify the fraction, dynamics, and function of homo-oligomers in single living cells in response to DNA damage.p53 is a stress-response transcription factor that orchestrates cell fate decisions such as cell-cycle arrest, senescence, and apoptosis. Tetramerization of p53 is required for its direct binding to DNA (3,4). Mutations in the p53 tetramerization domain (326-356 aa) lead to a reduction in, or loss of, its transcriptional activity in cells (5) and were shown to cause early cancer onset, known as Li-Fraumeni syndrome (6, 7).In in vitro studies, p53 first assembles into homo-dimers with a K d of ∼1 nM (8), and these dimers then come together in tetramers with a K d of ∼100 nM-1 μM (8-11). The K d of tetramerization in vitro can be lowered by specific posttranslational modifications (10-12). Based on these measurements and the estimated p53 concentration in cells of 140 nM (13), it has been proposed that p53 should be primarily dimeric in basal conditions and that it forms tetramers in stressed conditions (14). However, there is currently no direct experimental evidence for this in cells.We used...