Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding.

The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a one-dimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

Binding investigation between M2-1protein from hRSV and acetylated quercetin derivatives: 1H NMR, fluorescence spectroscopy, and molecular docking.

The human Respiratory Syncytial Virus (hRSV) is the main responsible for occurrences of respiratory diseases as pneumonia and bronchiolitis in children and elderly. M2-1 protein from hRSV is an important antitermination factor for transcription process that prevents the premature dissociation of the polymerase complex, making it a potential target for developing of inhibitors of the viral replication. The present study reports the interaction of the M2-1 tetramer with pera (Q1) and tetracetylated (Q2) quercetin derivatives, which were synthesized with the objective of generating stronger bioactive compounds against oxidation process. Fluorescence experiments showed binding constants of the M2-1/compounds complexes on order of 104M-1 with one ligand per monomeric unit, being the affinity of Q2 stronger than Q1. The thermodynamic analysis revealed values of ΔH>0 and ΔS>0, suggesting that hydrophobic interactions play a key role in the formation of the complexes. Molecular docking calculations indicated that binding sites for the compounds are in contact interfaces between globular and zinc finger domains of the monomers and that hydrogen bonds and stacking interactions are important contributions for stabilization of the complexes. Thus, the interaction of the acetylated quercetin derivatives in the RNA-binding sites of M2-1 makes these potential candidates for viral replication inhibitors.