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...

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious disease.