Role of Glutamine 17 of the Bovine Papillomavirus E5 Protein in Platelet-Derived Growth Factor β Receptor Activation and Cell Transformation

Abstract

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF β receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF β receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF β receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF β receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF β receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF β receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF β receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF β receptor.

Structural models of the bovine papillomavirus E5 protein

“…This is consistent with experimental evidence that substitution of Gln17 with hydrophobic residues destabilizes, but does not completely disrupt, the E5 dimer. 42 The residue with the largest energy contribution in both clusters is Tyr31. This tyrosine is wellpacked in the interface with other aromatic residues and is hydrogen-bonded to Glu36 of the opposite helix.…”

“…The observation that hydrogen bonding of Gln17 across the dimer interface stabilizes the E5 dimer is consistent with a role for Gln17 in dimerization (criterion iii) favoring cluster 2 as the structure of the E5 dimer. In particular, E5 dimer formation is reduced by replacing Gln17 with hydrophobic amino acids unable to form hydrogen bonds across the dimer interface (Meyer et al, 15 ; O. Klein et al, 42 ). Cluster 2 may also be favored energetically since the interaction energies calculated in Figure 3 do not reflect the unfavorable situation in cluster 1 where the polar glutamine side chains are exposed to a low dielectric environment and may not be in a position to form side-chain hydrogen bonds.…”

“…Recent studies systematically examining all possible position 17 mutants revealed a strong correlation between E5-PDGF-␤ receptor complex formation, receptor activation, and transformation for the various mutants in murine C127 cells. 42 Moreover, the identity of the amino acid at position 17 had marked effects on the extent of E5 dimerization, with hydrophobic substitutions in general allowing the least dimer formation.…”

The Bovine Papillomavirus E5 Protein Requires a Juxtamembrane Negative Charge for Activation of the Platelet-Derived Growth Factor β Receptor and Transformation of C127 Cells

“…Four absolutely conserved residues in the E5 protein are important for binding and activation of the PDGF ␤ receptor and for cell transformation: the transmembrane glutamine 17, the juxtamembrane aspartic acid 33, and the C-terminal cysteines 37 and 39, which are involved in homodimerization of the E5 protein ( Fig. 1) (8,10,14,15). In addition, the overall hydrophobicity of the central region of the E5 protein, but not the specific amino acid sequence, is critical for cell transformation (12,14).…”

“…1). These data have led to the proposal that the E5 protein and the PDGF ␤ receptor interact directly with one another and that two pairs of interacting residues, aspartate 33-lysine 499 and glutamine 17-threonine 513, stabilize the E5 protein-PDGF ␤ receptor complex (10,14,19,23).…”