Mechanism of phosphorylation dependent interactions of complete Retinoblastoma-AB pocket domain with its linker

Retinoblastoma protein (pRb) is a critical tumor suppressor, whose activity is regulated through phosphorylation of Serine/Threonine residues within unstructured linker regions. Due to the absence of high-resolution structures encompassing these linkers, many studies have indirectly examined the phosphorylation effects on pRb-protein interactions. Here, we present computational models of the full pRb pocket domain (pRbAB), incorporating the flexible linker (pRbPL) that connects boxes A and B. Three models, generated by ROBETTA and AlphaFold, were used to explore the mechanisms by which phosphorylation at Ser608 and Ser612 influence the interaction of pRbAB with E2F transactivation domain (E2FTA). All models revealed a short helix (residues 602–607), similar to that observed in a previously published crystal structure of pRbAB with shortened pRbPL linker and phosphomimetic mutation at Ser608, with E2FTA (PDB:4ELL), and proposed to competitively inhibit E2FTA binding. Interestingly, this helix formed irrespective of phosphorylation, indicating that additional factors influencing the interaction upon phosphorylation. In our models, the pRbPL linker displayed significant motion, especially near Ser608 and Ser612, facilitating phosphorylation, but without inducing large conformational changes in the pocket domains. Phosphorylation at Ser608 and Ser612 resulted in changes in multiple parameters including electrostatic properties, compactness and energy landscape of pRbAB constituting important mechanisms that promote E2F release. Additionally, recapitulating known R661W mutation in these models correctly showed reduced E2FTA binding, demonstrating the predictive power of these models to study the effects of missense mutations on the stability, conformation and interactions of pRbAB with E2FTA.