Phosphorylation-induced Conformational Changes in the Retinoblastoma Protein Inhibit E2F Transactivation Domain Binding*

Inactivation of the retinoblastoma protein (Rb) through phosphorylation is an important step in promoting cell cycle progression, and hyperphosphorylated Rb is commonly found in tumors. Rb phosphorylation prevents its association with the E2F transcription factor; however, the molecular basis for complex inhibition has not been established. We identify here the key phosphorylation events and conformational changes that occur in Rb to inhibit the specific association between the E2F transactivation domain (E2FTD) and the Rb pocket domain. Calorimetry assays demonstrate that phosphorylation of Rb reduces the affinity of E2FTD binding ∼250-fold and that phosphorylation at Ser608/Ser612 and Thr356/Thr373 is necessary and sufficient for this effect. An NMR assay identifies phosphorylation-driven conformational changes in Rb that directly inhibit E2FTD binding. We find that phosphorylation at Ser608/Ser612 promotes an intramolecular association between a conserved sequence in the flexible pocket linker and the pocket domain of Rb that occludes the E2FTD binding site. We also find that phosphorylation of Thr356/Thr373 inhibits E2FTD binding in a manner that requires the Rb N-terminal domain. Taken together, our results suggest two distinct mechanisms for how phosphorylation of Rb modulates association between E2FTD and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.