Long-range modulation of chain motions within the intrinsically disordered transactivation domain of tumor suppressor p53.
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ABSTRACT:
SUBMITTER: Lum JK
PROVIDER: S-EPMC3265989 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC2311362 | biostudies-literature
Project description:Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally 15N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination.
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Project description:Intrinsically disordered proteins (IDPs) are frequently associated with human diseases such as cancers, and about one-fourth of disease-associated missense mutations have been mapped into predicted disordered regions. Understanding how these mutations affect the structure-function relationship of IDPs is a formidable task that requires detailed characterization of the disordered conformational ensembles. Implicit solvent coupled with enhanced sampling has been proposed to provide a balance between accuracy and efficiency necessary for systematic and comparative assessments of the effects of mutations as well as post-translational modifications on IDP structure and interaction. Here, we utilize a recently developed replica exchange with guided annealing enhanced sampling technique to calculate well-converged atomistic conformational ensembles of the intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 and several cancer-associated mutants in implicit solvent. The simulations are critically assessed by quantitative comparisons with several types of experimental data that provide structural information on both secondary and tertiary levels. The results show that the calculated ensembles reproduce local structural features of wild-type p53-TAD and the effects of K24N mutation quantitatively. On the tertiary level, the simulated ensembles are overly compact, even though they appear to recapitulate the overall features of transient long-range contacts qualitatively. A key finding is that, while p53-TAD and its cancer mutants sample a similar set of conformational states, cancer mutants could introduce both local and long-range structural modulations to potentially perturb the balance of p53 binding to various regulatory proteins and further alter how this balance is regulated by multisite phosphorylation of p53-TAD. The current study clearly demonstrates the promise of atomistic simulations for detailed characterization of IDP conformations, and at the same time reveals important limitations in the current implicit solvent protein force field that must be sufficiently addressed for reliable description of long-range structural features of the disordered ensembles.
| S-EPMC4405366 | biostudies-literature
Project description:Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.
| S-EPMC8179352 | biostudies-literature
Project description:Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine ?-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric ?-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ?4.6 nm to ?1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.
| S-EPMC3183800 | biostudies-other
Project description:Intersegmental transfer (IST) is an important strategy in the target search used by sequence-specific DNA-binding proteins (DBPs), enabling DBPs to search for targets between multiple DNA strands without dissociation. We examined the IST of the tumor suppressor p53 using ensemble stopped-flow and single-molecule fluorescence measurements. The ensemble measurements demonstrated that p53 exhibits very fast IST, whose rate constant was ?108 M-1 s-1. To determine the domains of p53 responsible for IST, two mutants with deletions of one of its two DNA binding domains were generated. The mutant lacking the disordered C-terminal (CT) domain (the CoreTet mutant) abolished IST, whereas the mutant lacking the structured core domain (the TetCT mutant) maintained IST, clearly demonstrating the importance of the CT domain. Single-molecule fluorescence measurements further demonstrated the transfer of p53 between two tethered DNA strands. The pseudo-wild-type p53 and the TetCT mutant showed significant transfer efficiencies, whereas the transfer efficiency for the CoreTet mutant was zero. These results suggest that ultrafast IST might be promoted by four copies of the CT domain, by binding to two DNA strands simultaneously. Such ultrafast IST might be important to avoid nearby-bound DBPs during the target search process of p53 in nucleus.
| S-EPMC6101536 | biostudies-literature
Project description:Transcription factor cyclic Adenosine monophosphate response-element binding protein plays a critical role in the cyclic AMP response pathway via its intrinsically disordered kinase inducible transactivation domain (KID). KID is one of the most studied intrinsically disordered proteins (IDPs), although most previous studies focus on characterizing its disordered state structures. An interesting question that remains to be answered is how the order-disorder transition occurs at experimental conditions. Thanks to the newly developed IDP-specific force field ff14IDPSFF, the quality of conformer sampling for IDPs has been dramatically improved. In this study, molecular dynamics (MD) simulations were used to study the order-to-disorder transition kinetics of KID based on the good agreement with the experiment on its disordered-state properties. Specifically, we tested four force fields, ff99SBildn, ff99IDPs, ff14IDPSFF, and ff14IDPs in the simulations of KID and found that ff14IDPSFF can generate more diversified disordered conformers and also reproduce more accurate experimental secondary chemical shifts. Kinetics analysis of MD simulations demonstrates that the order-disorder transition of KID obeys the first-order kinetics, and the transition nucleus is I127/L128/L141. The possible transition pathways from the nucleus to the last folded residues were identified as I127-R125-L138-L141-S143-A145 and L128-R125-L138-L141-S143-A145 based on a residue-level dynamical network analysis. These computational studies not only provide testable prediction/hypothesis on the order-disorder transition of KID but also confirm that the ff14IDPSFF force field can be used to explore the correlation between the structure and function of IDPs.
| S-EPMC7156114 | biostudies-literature
Project description:Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1-10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 10(10) M(-1) s(-1), approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.
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Project description:CREB-binding protein is a multidomain transcriptional coactivator whose transcriptional adaptor zinc-binding 1 (TAZ1) domain mediates interactions with a number of intrinsically disordered transactivation domains (TADs), including the CREB-binding protein/p300-interacting transactivator with ED-rich tail, the hypoxia inducible factor 1α, p53, the signal transducer and activator of transcription 2, and the NF-κB p65 subunit. These five disordered TADs undergo partial disorder-to-order transitions upon binding TAZ1, forming fuzzy complexes with helical segments. Interestingly, they wrap around TAZ1 with different orientations and occupy the binding sites with various orders. To elucidate the microscopic molecular details of the binding processes of TADs with TAZ1, in this work, we carried out extensive molecular dynamics simulations using a coarse-grained topology-based model. After careful calibration of the models to reproduce the residual helical contents and binding affinities, our simulations were able to recapitulate the experimentally observed flexibility profiles. Although great differences exist in the complex structures, we found similarities between hypoxia inducible factor 1α and signal transducer and activator of transcription 2 as well as between CREB-binding protein/p300-interacting transactivator with ED-rich tail and NF-κB p65 subunit in the binding kinetics and binding thermodynamics. Although the origins of similarities and differences in the binding mechanisms remain unclear, our results provide some clues that indicate that binding of TADs to TAZ1 could be templated by the target as well as encoded by the TADs.
| S-EPMC6818164 | biostudies-literature
Project description:Intrinsically disordered regions (IDRs) of proteins are implicated in key macromolecular interactions. However, the molecular forces underlying IDR function within multicomponent assemblies remain elusive. By combining thermodynamic and structural data, we have discovered an allostery-based mechanism regulating the soluble core region of the nuclear pore complex (NPC) composed of nucleoporins Nup53, Nic96, and Nup157. We have identified distinct IDRs in Nup53 that are functionally coupled when binding to partner nucleoporins and karyopherins (Kaps) involved in NPC assembly and nucleocytoplasmic transport. We show that the Nup53·Kap121 complex forms an ensemble of structures that destabilize Nup53 hub interactions. Our study provides a molecular framework for understanding how disordered and folded domains communicate within macromolecular complexes.
| S-EPMC6881172 | biostudies-literature