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

Abstract

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 onedimensional 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.

Grb2 Y160F mutant mimics the wild-type monomeric state dynamics and the monomer-dimer equilibrium

“…Curiously, in contrast to the wild-type, these new conformations reveal that the SH3 domains move apart and do not interact with each other, rotating in opposite directions towards the SH2 domain. This result agrees with the multiple conformations in which both SH3 domains take different positions and orientations in relation to the SH2 domain [14], [17].…”

Understanding the Energy Landscape of Intrinsically Disordered Protein Ensembles

“…Previous studies with ELViM were carried out using strictly molecular dynamics trajectories, and they have proven useful in the investigation of molecular mechanisms, identifying transition-state ensembles, folding routes, and metastable states. The analyzed systems encompass ordered proteins, 39,49,55 knotted proteins, 51 disordered peptides and proteins, 50,54,56 and an RNA tetraloop. 53 In the present study, we have addressed IDP ensembles that were obtained by a variety of integrative experimental and computational methods.…”

Understanding the Energy Landscape of Intrinsically Disordered Protein Ensembles

“…Previous studies with ELViM were carried out using strictly molecular dynamics trajectories, and they have proven useful in the investigation of molecular mechanisms, identifying transition-state ensembles, folding routes, and metastable states. The analyzed systems encompass ordered proteins, ,, knotted proteins, disordered peptides and proteins, ,, and an RNA tetraloop . In the present study, we have addressed IDP ensembles that were obtained by a variety of integrative experimental and computational methods.…”