Using chemical shifts to generate structural ensembles for intrinsically disordered proteins with converged distributions of secondary structure.
ABSTRACT: A short segment of the disordered p53 transactivation domain (p53TAD) forms an amphipathic helix when bound to the E3 ubiquitin ligase, MDM2. In the unbound p53TAD, this short segment has transient helical secondary structure. Using a method that combines broad sampling of conformational space with re-weighting, it is shown that it is possible to generate multiple, independent structural ensembles that have highly similar secondary structure distributions for both p53TAD and a P27A mutant. Fractional amounts of transient helical secondary structure were found at the MDM2 binding site that are very similar to estimates based directly on experimental observations. Structures were identified in these ensembles containing segments that are highly similar to short p53 peptides bound to MDM2, even though the ensembles were re-weighted using unbound experimental data. Ensembles were generated using chemical shift data (alpha carbon only, or in combination with other chemical shifts) and cross-validated by predicting residual dipolar couplings. We think this ensemble generator could be used to predict the bound state structure of protein interaction sites in IDPs if there are detectable amounts of matching transient secondary structure in the unbound state.
Project description:The level of the p53 transcription factor is negatively regulated by the E3 ubiquitin ligase murine double-minute clone 2 (MDM2). The interaction between p53 and MDM2 is essential for the maintenance of genomic integrity for most eukaryotes. Previous structural studies revealed that MDM2 binds to p53 transactivation domain (p53TAD) from residues 17 to 29. The K24N mutation of p53TAD changes a lysine at position 24 to an asparagine. This mutation occurs naturally in the bovine family and is also found in a rare form of human gestational cancer called choriocarcinoma. In this study, we have investigated how the K24N mutation affects the affinity, structure, and dynamics of p53TAD binding to MDM2. Nuclear magnetic resonance studies of p53TAD show that the K24N mutant is more flexible and has less transient helical secondary structure than the wild type. Isothermal titration calorimetry measurements demonstrate that these changes in structure and dynamics do not significantly change the binding affinity for p53TAD-MDM2. Finally, free-energy perturbation and standard molecular dynamic simulations suggest the negligible affinity change is due to a compensating interaction energy between the K24N mutant and the MDM2 when it is bound. Overall, the data suggest that the K24N-MDM2 complex is able to, at least partly, compensate for an increase in the conformational entropy in unbound K24N with an increase in the bound-state electrostatic interaction energy.
Project description:The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3?5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.
Project description:The p53, p63, and p73 proteins belong to the p53 family of transcription factors, which play key roles in tumor suppression. Although the transactivation domains (TADs) of the p53 family are intrinsically disordered, these domains are commonly involved in the regulatory interactions with mouse double minute 2 (MDM2). In this study, we determined the solution structure of the p73TAD peptide in complex with MDM2 using NMR spectroscopy and biophysically characterized the interactions between the p53 family TAD peptides and MDM2. In combination with mutagenesis data, the complex structures revealed remarkably close mimicry of the MDM2 recognition mechanism among the p53 family TADs. Upon binding with MDM2, the intrinsically disordered p73TAD and p63TAD peptides adopt an amphipathic ?-helical conformation, which is similar to the conformation of p53TAD, although the ?-helical content induced by MDM2 binding varies. With isothermal titration calorimetry (ITC) and circular dichroism (CD) data, our biophysical characterization showed that p73TAD resembles p53TAD more closely than p63TAD in terms of helical stability, MDM2 binding affinity, and phosphorylation effects on MDM2 binding. Therefore, our structural information may be useful in establishing alternative anticancer strategies that exploit the activation of the p73 pathway against human tumors bearing p53 mutations.
Project description:We report the first experimental measurements of Ramachandran ?-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an ?-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the ?-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.
Project description:The interaction between the transactivation domain of p53 (p53TAD) and the N-terminal domain of MDM2 and MDMX plays an essential role for cell function. Mutations in these proteins have been implicated in many forms of cancer. The intrinsically disordered p53TAD contains two subdomains, TAD1 and TAD2. Using NMR spectroscopy, site-directed mutagenesis, and molecular dynamics simulations, we demonstrate that TAD2 directly interacts with MDM2, adopting transient structures that bind to the same hydrophobic pocket of MDM2 as TAD1. Our data show that binding of TAD1 and TAD2 to MDM2 is competitive, which is further supported by the observation that the interaction of TAD2 with MDM2 can be blocked by the small molecule inhibitor nutlin-3. Our data further indicate that TAD2 interacts with MDMX in a fashion very similar to MDM2. Because TAD2 is known to have transcriptional activity, the interaction of TAD2 with MDM2/MDMX may play a direct role in the inhibition of p53 transactivation.
Project description:Disordered regions and Intrinsically Disordered Proteins (IDPs) are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD), a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how post-translational modifications can regulate the function of IDPs.
Project description:Protein-protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.
Project description:MDM2 is a negative regulator of p53 activity and an important target for cancer therapeutics. The N-terminal lid region of MDM2 modulates interactions with p53 via competition for its binding cleft, exchanging slowly between docked and undocked conformations in the absence of p53. To better understand these dynamics, we constructed Markov State Models (MSMs) from large collections of unbiased simulation trajectories of apo-MDM2, and find strong evidence for diffuse, yet two-state folding and binding of the N-terminal region to the p53 receptor site. The MSM also identifies holo-like receptor conformations highly suitable for computational docking, despite initiating trajectories from closed-cleft receptor structures unsuitable for docking. Fixed-anchor docking studies using a test set of high-affinity small molecules and peptides show simulated receptor ensembles achieve docking successes comparable to cross-docking studies using crystal structures of receptors bound by alternative ligands. For p53, the best-scoring receptor structures have the N-terminal region lid region bound in a helical conformation mimicking the bound structure of p53, suggesting lid region association induces receptor conformations suitable for binding. These results suggest that MD?+?MSM approaches can sample binding-competent receptor conformations suitable for computational peptidomimetic design, and that inclusion of disordered regions may be essential to capturing the correct receptor dynamics.
Project description:The goal of this article is to reduce the complexity of the side chain search within docking problems. We apply six methods of generating side chain conformers to unbound protein structures and determine their ability of obtaining the bound conformation in small ensembles of conformers. Methods are evaluated in terms of the positions of side chain end groups. Results for 68 protein complexes yield two important observations. First, the end-group positions change less than 1 Å on association for over 60% of interface side chains. Thus, the unbound protein structure carries substantial information about the side chains in the bound state, and the inclusion of the unbound conformation into the ensemble of conformers is very beneficial. Second, considering each surface side chain separately in its protein environment, small ensembles of low-energy states include the bound conformation for a large fraction of side chains. In particular, the ensemble consisting of the unbound conformation and the two highest probability predicted conformers includes the bound conformer with an accuracy of 1 Å for 78% of interface side chains. As more than 60% of the interface side chains have only one conformer and many others only a few, these ensembles of low-energy states substantially reduce the complexity of side chain search in docking problems. This approach was already used for finding pockets in protein-protein interfaces that can bind small molecules to potentially disrupt protein-protein interactions. Side-chain search with the reduced search space will also be incorporated into protein docking algorithms.
Project description:Physicochemical description of numerous cell processes is fundamentally based on the energy landscapes of protein molecules involved. Although the whole energy landscape is difficult to reconstruct, increased attention to particular targets has provided enough structures for mapping functionally important subspaces associated with the unbound and bound protein structures. The subspace mapping produces a discrete representation of the landscape, further called energy spectrum. We compiled and characterized ensembles of bound and unbound conformations of six small proteins and explored their spectra in implicit solvent. First, the analysis of the unbound-to-bound changes points to conformational selection as the binding mechanism for four proteins. Second, results show that bound and unbound spectra often significantly overlap. Moreover, the larger the overlap the smaller the root mean square deviation (RMSD) between the bound and unbound conformational ensembles. Third, the center of the unbound spectrum has a higher energy than the center of the corresponding bound spectrum of the dimeric and multimeric states for most of the proteins. This suggests that the unbound states often have larger entropy than the bound states. Fourth, the exhaustively long minimization, making small intrarotamer adjustments (all-atom RMSD ? 0.7 Å), dramatically reduces the distance between the centers of the bound and unbound spectra as well as the spectra extent. It condenses unbound and bound energy levels into a thin layer at the bottom of the energy landscape with the energy spacing that varies between 0.8-4.6 and 3.5-10.5 kcal/mol for the unbound and bound states correspondingly. Finally, the analysis of protein energy fluctuations showed that protein vibrations itself can excite the interstate transitions, including the unbound-to-bound ones.