Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation

Abstract

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.transmembrane protein complex | oncogene | traptamer | BPV | blue native gel electrophoresis B ecause viruses modulate signaling nodes that control cell behavior and virus replication, the study of viral proteins has provided great insight into many aspects of cellular biochemistry and the cellular processes that regulate biological function. Thus, viral proteins have long been recognized as valuable tools to probe central problems in biology. A particularly interesting viral protein is the 44-residue E5 oncoprotein encoded by bovine papillomavirus type 1 (BPV). The BPV E5 protein and closely related E5 proteins of other fibropapillomaviruses are the shortest known, naturally occurring proteins with tumorigenic potential (1). The E5 protein is an extremely hydrophobic integral membrane protein located primarily in the membranes of the Golgi apparatus of transformed cells (2, 3). Biophysical studies in model membranes indicate that the E5 protein adopts a transmembrane orientation roughly perpendicular to the membrane surface (4-6). In essence, the E5 protein is a freestanding transmembrane domain (TMD), with a type II transmembrane orientation in which a short C-terminal segment protrudes into the lumen of the Golgi (2). In cells, detergent micelles, and model lipid bilayers, the E5 protein exists as a homodimer stabilized by disulfide bonds involving C-terminal cysteine residues (3-5, 7-10). Genetic studies showed that E5 dimerization is required for transforming activity, and a preferred orientation of the E5 dimer with a symmetric homodimer interface has been identified (7,9,11,12).The E5 protein transforms cells by activating the cellular platelet-derived growth factor (PDGF) β receptor (PDGFβR), although there may be additional minor, alternative transforming pathways as well (13). The PDGFβR is a receptor tyrosine kinase (RTK) with an extracellular domain that binds PDGF, a hydrophobic membrane-spanning segment, and a cytoplasmic domain with tyrosine kinase activity. The inactive PDGFβR is primarily monomeric in unstimulated cells, and PDGF binding indu...

Oral fibropapillomatosis and epidermal hyperplasia of the lip in newborn lambs associated with bovine Deltapapillomavirus

“…Indeed, immunoblotting studies detected a dimeric conformation of the viral E5 protein in some organs of these lambs. This suggests that E5 protein was present as a dimer which is known to be the biologically active conformation of the protein 32 . To the authors’ knowledge, BPVs have not been previously reported as a cause of clinical disease in sheep.…”

The human papillomavirus oncoproteins: a review of the host pathways targeted on the road to transformation

“…One critical function of BPV1 E5 is activation of the platelet-derived growth factor receptor (PDGFR), resulting in downstream mitogenic signalling, enhanced DNA synthesis and transformation [ 60, 61 ]. This occurs via direct binding of BPV1 E5 to the PDGFRβ isoform [ 62 ]. In contrast, there is no evidence for BPV4 E5 activation of growth-factor receptors; rather it disrupts the expression and activity of key cell-cycle regulators such as cyclin A and p27 KIP1 [ 63 ].…”

Bovine Papillomavirus Type 2 Infection Associated with Papillomatosis of the Amniotic Membrane in Water Buffaloes (Bubalus bubalis)

“…In addition, foci of squamous epithelium composed of numerous cell layers on the internal surface of the amnion have been found to form the so-called amniotic plaques that are apparently normal ill-defined structures, whose physiological function, if any, is unknown [28]. Usually, definitive amniotic plaques are flat in appearance and vary in size from 2 to 4 mm in diameter [28,37]; their height can be up to 8 mm [29]. They have been described in white-tailed deer [37], in cows [38], and in ewes [39,40].…”

“…Similar levels of total PDGFR were detected in both six papillomatous and two grossly normal placentas, whereas there was a statistically significant (p  0.05) increase of phosphorylated PDGFR in papillomatous placentas compared to the samples of grossly normal placentas (Figure 8). As it has been shown that E5 dimerization is required for transforming activity via phosphorylation of the PDGFβR [29], an immunoblotting investigation to detect total and phosphorylated PDGFβR was also performed. Similar levels of total PDGFβR were detected in both six papillomatous and two grossly normal placentas, whereas there was a statistically significant (p ≤ 0.05) increase of phosphorylated PDGFβR in papillomatous placentas compared to the samples of grossly normal placentas (Figure 8).…”

“…As it has been shown that E5 dimerization is required for transforming activity via phosphorylation of the PDGFR [29], an immunoblotting investigation to detect total and phosphorylated PDGFR was also performed. Similar levels of total PDGFR were detected in both six papillomatous and two grossly normal placentas, whereas there was a statistically significant (p  0.05) increase of phosphorylated PDGFR in papillomatous placentas compared to the samples of grossly normal placentas (Figure 8).…”