Association of Epha2 Receptor with the Membrane through its Second Fibronectin Domain: A Biophysical and Computational Study

Abstract

Eph receptors are cell surface type I transmembrane receptors involved in numerous cellular processes. Ligand-binding causes Eph clustering, a process that is, to a large extent, driven by the extracellular region of the receptor. The structural mechanisms that underly Eph clustering have recently been elucidated by crystal structure data for complete Eph ectodomains and their ligands. While it is understood that the clustering depends largely on the N-terminal domains of the extracellular region, little is known about the functions of the downstream domains that lead into the transmembrane helix. To address this issue, we have combined multiscale molecular dynamics (MD) simulations with biophysical assays to study the extracellular region of EphA2 upstream of the transmembrane helix, the fibronectin domain 2 (FN2). A number of novel insights emerged: (i) the FN2 domain can interact with the membrane, (ii) this domain preferentially interacts with anionic lipids, (iii) this preference is maintained through a positively charged surface of the protein, including K441 and R443. The study allows us, for the first time, to combine the recently emerged X-ray crystallography with NMR models of the transmembrane region. We present a full atomistic model of an Eph signalling cluster (extracellular regions and transmembrane regions) assembled at a model cell membrane composed of a lipid mixture. This work is a significant step in understanding the formation of Eph signalling clusters and paves the way towards understanding the assembly of full length Eph receptor platforms at the surface of cells.