Wordom is a versatile program for manipulation of molecular dynamics trajectories and efficient analysis of simulations. Original tools in Wordom include a procedure to evaluate significance of sampling for principal component analysis as well as modules for clustering multiple conformations and evaluation of order parameters for folding and aggregation. The program was developed with special emphasis on user-friendliness, effortless addition of new modules and efficient handling of large sets of trajectories.

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The p53 cancer mutation Y220C induces formation of a cavity on the protein's surface that can accommodate stabilizing small molecules. We combined fragment screening and molecular dynamics to assess the druggability of p53-Y220C and map ligand interaction sites within the mutational cavity. Elucidation of the binding mode of fragment hits by crystallography yielded a clear picture of how a drug might dock in the cavity. Simulations that solvate the protein with isopropanol found additional sites that extend the druggable surface. Moreover, structural observations and simulation revealed the dynamic landscape of the cavity, which improves our understanding of the impact of the mutation on p53 stability. This underpins the importance of considering flexibility of the cavity in screening for optimized ligands. Our findings provide a blueprint for the design of effective drugs that rescue p53-Y220C.

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66 -86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.natively disordered ͉ domain-domain interaction ͉ quaternary structure ͉ FRET ͉ time-resolved T he tumor suppressor p53 is a tetrameric, multidomain transcription factor that plays key roles in maintaining the integrity of the human genome and in DNA repair machinery (1, 2). p53 is a partly intrinsically disordered protein, containing two folded domains: the DNA-binding core domain (CD; residues 94-294) and the tetramerization domain (TetD; residues 323-360) (3, 4). The intrinsically disordered N-terminal domain (NTD; residues 1-94) and C-terminal domain (CTD; residues 360-393) (5, 6) mediate interactions with several proteins such as p300/CBP, MDM2, 14-3-3, and S100 family that in turn regulate the activity of p53. Moreover, the NTD and CTD are the target sites of numerous posttranslational modificiations that modulate the activity of p53.High-resolution structures of the CD and the TetD have been solved by using X-ray crystallography and NMR spectroscopy (3,4,7,8). But, the intrinsic instability and the presence of highly disordered regions in p53 have impeded the application of conventional structural studies on full-length p53 (flp53). A combination of NMR spectroscopy and small-angle X-ray scattering (SAXS) in solution with electron microscopy on immobilized samples was recently used to solve the quaternary structures of a mutationally stabilized human flp53 and its DNA complex (...

Extensive molecular dynamics simulations reveal that the interactions between proteins and poly(ethylene glycol) (PEG) can be described in terms of the surface composition of the proteins. PEG molecules accumulate around non-polar residues while avoiding the polar ones. A solvent-accessible-surface-area model of protein adsorption accurately fits a large set of data on the composition of the protein corona of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles recently obtained by label-free proteomic mass spectrometry.

In ⌽-value analysis, the effects of mutations on the folding kinetics are compared with the corresponding effects on thermodynamic stability to investigate the structure of the protein-folding transition state (TS). Here, molecular dynamics (MD) simulations (totaling 0.65 ms) have been performed for a large set of single-point mutants of a 20-residue three-stranded antiparallel ␤-sheet peptide. Between 57 and 120 folding events were sampled at near equilibrium for each mutant, allowing for accurate estimates of folding͞unfolding rates and stability changes. The ⌽ values calculated from folding and unfolding rates extracted from the MD trajectories are reliable if the stability loss upon mutation is larger than Ϸ0.6 kcal͞mol, which is observed for 8 of the 32 single-point mutants. The same heterogeneity of the TS of the wild type was found in the mutated peptides, showing two possible pathways for folding. Single-point mutations can induce significant TS shifts not always detected by ⌽-value analysis. Specific nonnative interactions at the TS were observed in most of the peptides studied here. The interpretation of ⌽ values based on the ratio of atomic contacts at the TS over the native state, which has been used in the past in MD and Monte Carlo simulations, is in agreement with the TS structures of wild-type peptide. However, ⌽ values tend to overestimate the nativeness of the TS ensemble, when interpreted neglecting the nonnative interactions. ⌽ values are usually interpreted in terms of native contacts (4). This description has been successfully used to obtain sets of conformations from the TS ensemble of several proteins (5-9) and to bias molecular dynamics (MD) trajectories toward the TS (10). On the other hand, specific nonnative interactions may be formed at both the TS and denatured-state ensemble and lead to a wrong picture of TS if not taken into account (11). Furthermore, different experimental conditions or mutations may determine detectable changes in the TS structure, showing the presence of parallel pathways (12, 13) and, thus, a heterogeneous TS. In addition, the ensemble average associated with the use of certain folding observables, like the degree of tryptophan burial, may disguise the presence of multiple folding pathways and folding intermediates (14). Namely, a recent study (15) suggests that not all conformations obtained in MD simulations by using ⌽ values as restraints on a subset of the native contacts belong to the TS.The TS structures can be identified by MD simulations through the calculation of their folding probability P fold (16), i.e., the probability that a trajectory started from a given structure reaches the folded state before unfolding. The concept of P fold calculation was first introduced in a method for determining transmission coefficients, starting from a known TS (17), and used to identify TSs of simple conformational changes (e.g., tyrosine ring flips) (18). The approach has recently been used to study the otherwise very elusive folding TS by atomistic Monte Carlo off...