Protein-protein interactions in epidermal growth factor receptors through molecular dynamics

Resumen

The family of epidermal growth factor receptors (EGFR) is composed by four members: EGFR/ErbB1, ErbB2, ErbB3 and ErbB4. They are associated with a number of biological processes and are becoming increasingly recognized as important therapeutic targets against cancer. In this thesis, some models, based on homology, there are provided for the extracellular domains (ectodomain, ECD) of ErbB3 and ErbB4 in their active conformations, including a heregulin ligand, followed by further refinement of the models by molecular dynamics (MD) simulations at atomistic (AA) resolution. A model built for ErbB2 based on crystallographic information allowed an analysis of the common features observed among members of the family, namely, the periscope movement of the dimerization arm and the hinge displacement of subdomain IV. In addition, a refined model for the interaction of the ECD corresponding to a ErbB2/ErbB3 heterodimer is given. This heterodimer is widely recognized to have a high impact in cancer development. The ErbB2 receptor is a transmembrane oncoprotein that is over expressed in breast cancer. A successful therapeutic treatment is a monoclonal antibody called Trastuzumab which interacts with the ErbB2 extracellular domain (ECD-ErbB2). A better understanding of the detailed structure of the receptor-antibody interaction is indeed of prime interest for the design of more effective anticancer therapies. To analyze the flexibility of the complex ECD-ErbB2/Trastuzumab, a multi-nanosecond MD simulation together with an analysis of fluctuations through a principal component analysis (PCA) of this system, was carried out. For validating the simulations, a detailed analysis of the variable antibody domain interactions with the extracellular subdomain IV of ErbB2 was performed. This structure has been statically elucidated by X-ray crystallography. Indeed, the simulation results are in excellent agreement with the available experimental information. The PCA shows collective fluctuations resulting in a hinge motion in which subdomain II and constant domain (C_H) approach each other. This movement is likely stabilized by the formation of H-bonds and salt bridge interactions between residues of the dimerization arm in the subdomain II and the antibody Trastuzumab residues located in the C_H domain. Finally, the flexibility of the molecular dynamics model in relation with the static X-ray structure was discussed. A movement of the antibody towards the dimerization domain of the ErbB2 receptor is reported for the first time. This finding could have relevant consequences on the biological action of the monoclonal antibody. In addition to the above mentioned pure atomistic models, a mapping from AA to coarse-grained (CG) resolution has been validated. In this manner AA MD on ECD-ErbB2/Trastuzumab-Fab has been used to compare with CG MD simulations. Specifically, the CG Martini force field has been compared with the AA OPLS representation. The conformational flexibility and interactions between the antibody and the receptor have been analyzed. In this thesis the following parameters have been tested in the MD algorithms to carry out the Martini simulations: the non-bonded interactions methods to calculate the electrostatic interactions, the value of the neighbor lists cut-off radius (r_list) and the Elastic Network method. The results show that when used in MD simulations domElNeDyn models, PME and an r_list of 1.4 nm, are comparable to the AA protein models. The results shed light to validate the Martini force field in the protein--protein interactions and towards protein prediction structure. Therefore, CG MD simulations have been applied to study the influence of the Trastuzumab monoclonal antibody on the structure and dynamics of the full-length ErbB2 receptor dimer, including the lipid bilayer. The usage of CG models to study such complexes is almost mandatory, at present, due to the large size of the whole system. The Martini model performs satisfactorily well, giving results well-matched with those obtained by AA models as well as with the experimental information existing on homologous receptors. For example, the ecto and intracellular domains approach the bilayer surface in both the monomer and dimer cases. The Trastuzumab-Fab hinders the interaction of the receptors with the lipid bilayer. Another interesting effect of the antibody is the disruption of the antiparallel arrangement of the juxtamembrane segments in the dimer case. These findings might help to understand the effect of the antibody on the receptor bioactivity.