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...

Intrinsically disordered proteins (IDPs) are predicted to represent a significant fraction of the human genome, and the development of meaningful molecular descriptions of these proteins remains a key challenge for contemporary structural biology. In order to describe the conformational behavior of IDPs, a molecular representation of the disordered state based on diverse sources of structural data that often exhibit complex and very different averaging behavior is required. In this study, we propose a combination of paramagnetic relaxation enhancements (PREs) and residual dipolar couplings (RDCs) to define both long-range and local structural features of IDPs in solution. We demonstrate that ASTEROIDS, an ensemble selection algorithm, faithfully reproduces intramolecular contacts, even in the presence of highly diffuse, ill-defined target interactions. We also show that explicit modeling of spin-label mobility significantly improves the reproduction of experimental PRE data, even in the case of highly disordered proteins. Prediction of the effects of transient long-range contacts on RDC profiles reveals that weak intramolecular interactions can induce a severe distortion of the profiles that compromises the description of local conformational sampling if it is not correctly taken into account. We have developed a solution to this problem that involves efficiently combining RDC and PRE data to simultaneously determine long-range and local structure in highly flexible proteins. This combined analysis is shown to be essential for the accurate interpretation of experimental data from R-synuclein, an important IDP involved in human neurodegenerative disease, confirming the presence of long-range order between distant regions in the protein.