Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain

Abstract

Proteins with intrinsically disordered domains are implicated in a vast range of biological processes, especially in cell signaling and regulation. Having solved the quaternary structure of the folded domains in the tumor suppressor p53 by a multidisciplinary approach, we have now determined the average ensemble structure of the intrinsically disordered N-terminal transactivation domain (TAD) by using residual dipolar couplings (RDCs) from NMR spectroscopy and small-angle x-ray scattering (SAXS). Remarkably, not only were we able to measure RDCs of the isolated TAD, but we were also able to do so for the TAD in both the full-length tetrameric p53 protein and in its complex with a specific DNA response element. We determined the orientation of the TAD ensemble relative to the core domain, found that the TAD was stiffer in the proline-rich region (residues 64 -92), which has a tendency to adopt a polyproline II (PPII) structure, and projected the TAD away from the core. We located the TAD in SAXS experiments on a complex between tetrameric p53 and four Taz2 domains that bind tightly to the TAD (residues 1-57) and acted as ''reporters.'' The p53-Taz2 complex was an extended cross-shaped structure. The quality of the SAXS data enabled us to model the disordered termini and the folded domains in the complex with DNA. The core domains enveloped the response element in the center of the molecule, with the Taz2-bound TADs projecting outward from the core.hybrid methods ͉ natively unfolded ͉ protein ͉ residual dipolar coupling ͉ small-angle x-ray scattering T he tumor suppressor p53 is a multifunctional protein that plays vital roles in maintaining the integrity of the human genome, controlling apoptosis, cell-cycle arrest, and DNA repair (1). p53 is a homotetramer, with folded tetramerization and core domains that are linked together and flanked by intrinsically disordered (or natively unfolded) domains at the N and C termini (1, 2). As such, with 37% of its structure intrinsically disordered, p53 is typical of the structural content of the human proteome. More than 30% of eukaryotic genomes encode contiguous unfolded regions longer than 30 aa in length, and up to 80% in cancer-associated proteins (3). This new class of intrinsically disordered proteins (IDPs) is involved in a vast range of cellular processes, including molecular recognition, transcription and transposition, packaging, repair and replication, as well as signaling, cell cycle control, multiprotein complex assembly, and endocytosis. Many partly or fully disordered proteins undergo conformational transitions to folded forms only on interaction with a target ligand (4). An intrinsically disordered domain is possibly an essential structural feature that facilitates promiscuous binding to many partner proteins and is also readily accessible for posttranslational modification that modulates binding.Solving the structures of proteins with intrinsically disordered domains now represents a major stumbling block in relating structure and biological function. Class...

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

“…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)(5)(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 the subunits of the TET domain do not oligomerize (7,8).…”

“…Proteins were expressed as His-lipoyl domain fusions [full-length p53, p53C (94-296) and (89-296), TET L348A (325-355), p53 TAD-PRR-C (1-312), p53C-TET (96-355), and alanine point mutants of the proteins] were purified by combination of Ni 2þ -, heparin-affinity, and gel-filtration chromatography (6,22,27,36). Briefly: Cell lysis was performed using high-pressure homogenizer in a lysis buffer containing 25 mM K-phosphate, 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, pH ¼ 8.0.…”

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

“…Resonance assignments for WT human p53TAD were previously reported 12 and Bruker 750 MHz spectrometers equipped with pulse field Z-axis gradient cold probes, in 50 mM sodium phosphate buffer, 100 NaCl, 1 mM EDTA, 0.02% sodium azide and 2 mM DTT, at pH 6.8 (10% D 2 O). For HNCA and HNCACB experiments, data were acquired in 1 H, 13 C and 15 N dimensions using sweep widths of 13,020.8 (t 3 ) × 6,032.7 (t 2 ) × 3,000 (t 1 ) Hz and 1,024 (t 3 ) × 128 (t 2 ) × 32 (t 1 ) complex data points. The sweep widths and complex points for 2D HSQC experiments were 9,689.9 (t 2 ) × 2,500 (t 1 ) Hz and 1,024 (t All NMR spectra were processed with nmrPipe and analyzed in nmrView 26 .…”

“…In its free form, p53TAD exists in equilibrium between disordered and partially helical conformations [11][12][13] , whereas residues 19-25 form a stable amphipathic α-helix in the Mdm2 complex 14 (Fig. 1a).…”

Structure of the transition state for the binding of c-Myb and KIX highlights an unexpected order for a disordered system

“…From a thermodynamic perspective, it has been suggested that destabilization of the native protein structure, leading to its unfolding, lowers the affinity of a protein for its ligand, without necessarily abolishing specificity (4)(5)(6). Furthermore, the highly dynamic nature of IDPs was suggested to promote alternative binding of the same disordered segments to different partners, the so-called "moonlighting effect" (7,8), increasing the repertoire of activities. An interesting mechanistic model, proposed by Wolynes and coworkers (9), suggests that the IDPs display an increased capture radius to recruit and bind partners.…”