Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling.

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53's C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53's C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

Encapsulation of cannabidiol in oil-in-water nanoemulsions and nanoemulsion-filled hydrogels: A structure and biological assessment study.

HYPOTHESIS: Lipophilic cannabidiol can be solubilized in oil-in water nanoemulsions, which can then be impregnated into chitosan hydrogels forming another colloidal system that will facilitate cannabidiol's release. The delivery from both systems was compared, alongside structural and biological studies, to clarify the effect of the two carriers' structure on the release and toxicity of the systems. EXPERIMENTS: Oil-in-water nanoemulsions (NEs) and the respective nanoemulsion-filled chitosan hydrogels (NE/HGs) were formulated as carriers of cannabidiol (CBD). Size, polydispersity and stability of the NEs were evaluated and then membrane dynamics, shape and structure of both systems were investigated with EPR spin probing, SAXS and microscopy. Biocompatibility of the colloidal delivery systems was evaluated through cytotoxicity tests over normal human skin fibroblasts. An ex vivo permeation protocol using porcine ear skin was implemented to assess the release of CBD and its penetration through the skin. FINDINGS: Incorporation of the NEs in chitosan hydrogels does not significantly affect their structural properties as evidenced through SAXS, EPR and confocal microscopy. These findings indicate the successful development of a novel nanocarrier that preserves the NE structure with the CBD remaining encapsulated in the oil core while providing new rheological properties advantageous over NEs. Moreover, NE/HGs proved to be more efficient as a carrier for the release of CBD. Cell viability assessment revealed high biocompatibility of the proposed colloids.

Explicit treatment of water molecules in data-driven protein-protein docking: the solvated HADDOCKing approach.

Water molecules are active components in, literally, every biochemical event, forming hydrogen bonds, filling cavities, and mediating interactions with other (bio)molecules. Therefore, solvent drastically affects the kinetics and thermodynamics of numerous cellular events, including protein-protein interactions. While docking techniques are becoming successful in predicting the three-dimensional structure of protein-protein complexes, they are still limited in accounting explicitly for water in the binding process. HADDOCK is one of the few programs so far that can explicitly deal with water molecules during docking. Its solvated docking protocol starts from hydrated molecules, and a fraction of the interfacial water is subsequently removed from the docked models in a biased Monte Carlo procedure. The Monte Carlo-based removal is based on interfacial amino acid-water contact propensities derived from a dataset of high-resolution crystal structures of protein-protein complexes. In this chapter, this solvated docking protocol is described and associated methodological aspects are illustrated through an application example. It is shown that, although docking results do not always improve when the solvated docking protocol is applied, scoring is improved and the positions of buried water molecules in an interface are correctly predicted. Therefore, by identifying important water molecules, solvated docking can aid the development of novel inhibitors of protein-protein complexes that might be better accommodated at an interface.

Strengths and weaknesses of data-driven docking in critical assessment of prediction of interactions.

The recent CAPRI rounds have introduced new docking challenges in the form of protein-RNA complexes, multiple alternative interfaces, and an unprecedented number of targets for which homology modeling was required. We present here the performance of HADDOCK and its web server in the CAPRI experiment and discuss the strengths and weaknesses of data-driven docking. HADDOCK was successful for 6 out of 9 complexes (6 out of 11 targets) and accurately predicted the individual interfaces for two more complexes. The HADDOCK server, which is the first allowing the simultaneous docking of generic multi-body complexes, was successful in 4 out of 7 complexes for which it participated. In the scoring experiment, we predicted the highest number of targets of any group. The main weakness of data-driven docking revealed from these last CAPRI results is its vulnerability for incorrect experimental data related to the interface or the stoichiometry of the complex. At the same time, the use of experimental and/or predicted information is also the strength of our approach as evidenced for those targets for which accurate experimental information was available (e.g., the 10 three-stars predictions for T40!). Even when the models show a wrong orientation, the individual interfaces are generally well predicted with an average coverage of 60% ± 26% over all targets. This makes data-driven docking particularly valuable in a biological context to guide experimental studies like, for example, targeted mutagenesis.