Structural Conservation and E2F Binding Specificity within the Retinoblastoma Pocket Protein Family

The human pocket proteins retinoblastoma (Rb), p107, and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie the reason that Rb is a uniquely potent tumor suppressor. It has been proposed that distinct pocket protein activities are mediated by their different E2F transcription factor binding partners. In humans, Rb binds E2F1–E2F5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin-dependent kinase on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens the E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation. [Display omitted] •Pocket proteins (Rb, p107, and p130) and E2F transcription factors regulate cell-cycle-dependent gene expression.•Specific interactions between these proteins are due to affinity differences between the E2F transactivation domain and the pocket domain.•Mutating key interacting residues in the pocket domain of p107 results in a protein that binds E2F2-like Rb and regulates its transcription activity in cells.•Although phosphorylation of p107 is inefficient in vitro compared to Rb, analogous modifications inhibit E2F binding.