Structural Investigations of the Transmembrane Segment of the PDGF Receptor Beta ant the Oncoprotein E5 by Circular Dichroism and NMR

Abstract

The Platelet-derived growth factor receptor β (PDGFR) is a member of the receptor-tyrosine-kinase family involved in development. The receptor is activated when the natural ligand PDGF binds simultaneously to the ectodomains of two receptors, resulting in spatial proximity and subsequent cross-activation of the cytosolic kinase domains1). The receptor can also be activated independently of the natural ligand by binding of the small E5 oncoprotein from bovine papillomavirus2). E5 is a small membrane protein of only 44 amino acids and it is thought to manipulate the function of the receptor by specific helix-helix-contacts to the transmembrane of the receptor which then result in sustained receptor activation and cell transformation.To elucidate the helix-helix-interactions in receptor complex, we investigate the structure and membrane alignment of both proteins, first for each protein and later in the heterotetrameric four-helix-bundle. For the structural analysis we combined circular dichroism (CD) spectroscopy and liquid-state NMR to determine the secondary structure. Oriented CD and solid-state NMR were used to resolve the orientation of both proteins in their native environment. Therefore the E5 protein and the transmembrane domain of the receptor (PDGFRTMD) were 15N-isotope labelled by bacterial protein expression and reconstituted in detergent micelles and in oriented lipid bilayers. By CD and liquid-state NMR we found that both proteins are predominantly α-helical in detergent micelles and in lipid bilayers3,4). Furthermore, high resolution NMR measurements showed that PDGFRTMD forms a left-handed coiled-coil structure. A complementary OCD and solid-state NMR analysis of E5 and PDGFRTMD reconstituted in different lipid bilayers showed that the orientation of both proteins depends on the bilayer thickness, where both proteins were more tilted in thin membranes and less tilted in thick membranes.